2-(azaindol-2-yl) benz imidazoles as PAD4 inhibitors

ABSTRACT

Compounds of formula (I) wherein; R 1  is hydrogen or C 1-6 alkyl; R 2  is hydrogen, C 1-6 alkyl, perhalomethylC 0-5 alkyl-O—, or C 1-6 alkoxy; R 3  is hydrogen, C 1-6 alkyl, or C 1-6 alkoxyC 1-6 alkyl; R 4  is hydrogen, C 1-6 alkyl, perhalomethylC 1-6 alkyl; or unsubstituted C 3-6 cycloalkylC 1-6 alkyl; A is C—R 5  or N; B is C—R 6  or N; D is C—R 7  or N; with the proviso that at least one of A, B, and D, is N; R 5  is hydrogen or C 1-6 alkyl; R 6  is hydrogen or C 1-6 alkyl; R 7  is hydrogen, C 1-6 alkyl, C 1-6 alkoxy, or hydroxy; R 8  is hydrogen or C 1-6 alkyl, with the proviso that one of R 4  and R 8  is hydrogen; R 9  is hydrogen or hydroxy; R 10  is hydrogen or C 1-6 alkyl; and salts thereof are PAD4 inhibitors and may be useful in the treatment of various disorders, for example rheumatoid arthritis, vasculitis, systemic lupus erythematosus, ulcerative colitis, cancer, cystic fibrosis, asthma, cutaneous lupus erythematosis, and psoriasis.

FIELD OF THE INVENTION

The present invention is directed to certain novel compounds which areinhibitors of PAD4, processes for their preparation, pharmaceuticalcompositions comprising the compounds, and the use of the compounds orthe compositions in the treatment of various disorders. Compounds whichinhibit PAD4 may be useful in the treatment of various disorders, forexample rheumatoid arthritis, vasculitis, systemic lupus erythematosus,ulcerative colitis, cancer, cystic fibrosis, asthma, cutaneous lupuserythematosis, and psoriasis.

BACKGROUND OF THE INVENTION

PAD4 is a member of the peptidylarginine deiminase (PAD) family ofenzymes capable of catalysing the citrullination of arginine intocitrulline within peptide sequences. PAD4 is responsible for thedeimination or citrullination of a variety of proteins in vitro and invivo, with consequences of diverse functional responses in a variety ofdiseases (Jones J. E. et al, Curr. Opin. Drug Discov. Devel., 12(5),(2009), 616-627). Examples of exemplar diseases include rheumatoidarthritis, diseases with neutrophilic contributions to pathogenesis (forexample vasculitis, systemic lupus erythematosus, ulcerative colitis) inaddition to oncology indications. PAD4 inhibitors may also have widerapplicability as tools and therapeutics for human disease throughepigenetic mechanisms.

Inhibitors of PAD4 may have utility against Rheumatoid Arthritis (RA).RA is an auto-immune disease affecting approximately 1% of thepopulation (Wegner N. et al, Immunol. Rev., 233(1) (2010), 34-54). It ischaracterised by inflammation of articular joints leading todebilitating destruction of bone and cartilage. A weak geneticassociation between PAD4 polymorphisms and susceptibility to RA has beensuggested, albeit inconsistently, in a number of population studies(Kochi Y. et al, Ann. Rheum. Dis., 70, (2011), 512-515). PAD4 (alongwith family member PAD2) has been detected in synovial tissue where itis responsible for the deimination of a variety of joint proteins. Thisprocess is presumed to lead to a break of tolerance to, and initiationof immune responses to, citrullinated substrates such as fibrinogen,vimentin and collagen in RA joints. These anti-citrullinated proteinantibodies (ACPA) contribute to disease pathogenesis and may also beused as a diagnostic test for RA (e.g. the commercially available CCP2or cyclic citrullinated protein 2 test). In addition, increasedcitrullination may also offer additional direct contributions to diseasepathogenesis through its ability to affect directly the function ofseveral joint and inflammatory mediators (e.g. fibrinogen,anti-thrombin, multiple chemokines). In a smaller subset of RA patients,anti-PAD4 antibodies can be measured and may correlate with a moreerosive form of the disease.

PAD4 inhibitors may also be useful for the reduction of pathologicalneutrophil activity in a variety of diseases. Studies suggest that theprocess of Neutrophil Extracellular Trap (NET) formation, an innatedefence mechanism by which neutrophils are able to immobilise and killpathogens, is associated with histone citrulllination and is deficientin PAD4 knockout mice (Neeli I. et al, J. Immunol., 180, (2008),1895-1902 and Li P. et al, J. Exp. Med., 207(9), (2010), 1853-1862).PAD4 inhibitors may therefore have applicability for diseases where NETformation in tissues contributes to local injury and disease pathology.Such diseases include, but are not limited to, small vessel vasculitis(Kessenbrock K. et al, Nat. Med., 15(6), (2009), 623-625), systemiclupus erythematosus (Hakkim A. et al, Proc. Natl. Acad. Sci. USA,107(21), (2010), 9813-9818 and Villanueva E. et al, J. Immunol., 187(1),(2011), 538-52), ulcerative colitis (Savchenko A. et al, Pathol. Int.,61(5), (2011), 290-7), cystic fibrosis, asthma (Dworski R. et al, J.Allergy Clin. Immunol., 127(5), (2011), 1260-6), deep vein thrombosis(Fuchs T. et al, Proc. Natl. Acad. Sci. USA, 107(36), (2010), 15880-5),periodontitis (Vitkov L. et al, Ultrastructural Pathol., 34(1), (2010),25-30), sepsis (Clark S. R. et al, Nat. Med., 13(4), (2007), 463-9),appendicitis (Brinkmann V. et al, Science, 303, (2004), 1532-5), andstroke. In addition, there is evidence that NETs may contribute topathology in diseases affecting the skin, eg in cutaneous lupuserythematosis (Villanueva E. et al, J. Immunol., 187(1), (2011), 538-52)and psoriasis (Lin A. M. et al., J. Immunol., 187(1), (2011), 490-500),so a PAD4 inhibitor may show benefit to tackle NET skin diseases, whenadministered by a systemic or cutaneous route. PAD4 inhibitors mayaffect additional functions within neutrophils and have widerapplicability to neutrophilic diseases.

Studies have demonstrated efficacy of tool PAD inhibitors (for examplechloro-amidine) in a number of animal models of disease, includingcollagen-induced arthritis (Willis V. C. et al, J. Immunol., 186(7),(2011), 4396-4404), dextran sulfate sodium (DSS)-induced experimentalcolitis (Chumanevich A. A. et al, Am. J. Physiol. Gastrointest. LiverPhysiol., 300(6), (2011), G929-G938), spinal cord repair (Lange S. etal, Dev. Biol., 355(2), (2011), 205-14), and experimental autoimmuneencephalomyelitis (EAE). The DSS colitis report also demonstrates thatchloro-amidine drives apoptosis of inflammatory cells both in vitro andin vivo, suggesting that PAD4 inhibitors may be effective more generallyin widespread inflammatory diseases.

PAD4 inhibitors may also be useful in the treatment of cancers (Slack.J. L. et al, Cell. Mol. Life Sci., 68(4), (2011), 709-720).Over-expression of PAD4 has been demonstrated in numerous cancers (ChangX. et al, BMC Cancer, 9, (2009), 40). An anti-proliferative role hasbeen suggested for PAD4 inhibitors from the observation that PAD4citrullinates arginine residues in histones at the promoters ofp53-target genes such as p21, which are involved in cell cycle arrestand induction of apoptosis (Li P. et al, Mol. Cell Biol., 28(15),(2008), 4745-4758).

The aforementioned role of PAD4 in deiminating arginine residues inhistones may be indicative of a role for PAD4 in epigenetic regulationof gene expression. PAD4 is the primary PAD family member observed to beresident in the nucleus as well as the cytoplasm. Early evidence thatPAD4 may act as a histone demethyliminase as well as a deiminase isinconsistent and unproven. However, it may reduce histone argininemethylation (and hence epigenetic regulation associated with this mark)indirectly via depletion of available arginine residues by conversion tocitrulline. PAD4 inhibitors may therefore be useful as epigenetic toolsor therapeutics for affecting expression of varied target genes inadditional disease settings. Through such mechanisms, PAD4 inhibitorsmay also be effective in controlling citrullination levels in stem cellsand may therefore therapeutically affect the pluripotency status anddifferentiation potential of diverse stem cells including, but notlimited to, embryonic stem cells, neural stem cells, haematopoietic stemcells and cancer stem cells.

SUMMARY OF THE INVENTION

The invention is directed to compounds of formula (I):

wherein R₁, R₂, R₃, R₄, A, B, D, R₈, R₉, and R₁₀ are as defined below;and salts thereof.

Certain compounds of the invention have been shown to be PAD4 inhibitorsand may also show enhanced selectivity for PAD4 with respect to PAD2.For example, certain compounds of the invention indicate 1000-foldselectivity for PAD4 inhibition over PAD2 inhibition. Compounds whichinhibit PAD4 may be useful in the treatment of various disorders, forexample rheumatoid arthritis, vasculitis, systemic lupus erythematosus,ulcerative colitis, cancer, cystic fibrosis, asthma, cutaneous lupuserythematosis, and psoriasis. Accordingly, the invention is furtherdirected to pharmaceutical compositions comprising a compound of formula(I), or a pharmaceutically acceptable salt thereof. The invention isstill further directed to methods of treatment of disorders associatedtherewith using a compound of formula (I) or a pharmaceuticallyacceptable salt thereof, or a pharmaceutical composition comprising acompound of formula (I) or a pharmaceutically acceptable salt thereof.The invention is yet further directed towards processes for thepreparation of the compounds of the invention.

DETAILED DESCRIPTION OF THE INVENTION

In a first aspect, there are provided compounds of formula (I):

wherein;

R₁ is hydrogen or C₁₋₆alkyl;

R₂ is hydrogen, C₁₋₆alkyl, perhalomethylC₀₋₅alkyl-O—, or C₁₋₆alkoxy;

R₃ is hydrogen, C₁₋₆alkyl, or C₁₋₆alkoxyC₁₋₆alkyl;

R₄ is hydrogen, C₁₋₆alkyl, perhalomethylC₁₋₆alkyl; or unsubstitutedC₃₋₆cycloalkylC₁₋₆alkyl;

A is C—R₅ or N;

B is C—R₆ or N;

D is C—R₇ or N;

with the proviso that at least one of A, B, and D, is N;

R₅ is hydrogen or C₁₋₆alkyl;

R₆ is hydrogen or C₁₋₆alkyl;

R₇ is hydrogen, C₁₋₆alkyl, C₁₋₆alkoxy, or hydroxy;

R₈ is hydrogen or C₁₋₆alkyl, with the proviso that one of R₄ and R₈ ishydrogen;

R₉ is hydrogen or hydroxy;

R₁₀ is hydrogen or C₁₋₆alkyl;

and salts thereof.

In one embodiment, R₁ is hydrogen.

In one embodiment, R₁ is C₁₋₆alkyl.

In one embodiment, R₂ is hydrogen or C₁₋₆alkoxy.

In one embodiment, R₂ is C₁₋₆alkoxy.

In one embodiment, R₂ is perhalomethylC₀₋₅alkyl-O—.

In one embodiment, R₂ is trifluoromethoxy.

In one embodiment, R₃ is hydrogen.

In one embodiment, R₃ is C₁₋₆alkoxyC₁₋₆alkyl.

In one embodiment, R₃ is C₁₋₆alkyl.

In one embodiment, R₄ is C₁₋₆alkyl, unsubstitutedC₃₋₆cycloalkylC₁₋₆alkyl, or perhalomethylC₁₋₆alkyl.

In one embodiment, R₄ is C₁₋₆alkyl, unsubstitutedC₃₋₆cycloalkylC₁₋₆alkyl, or perfluoromethylC₁₋₆alkyl.

In one embodiment, R₅ is hydrogen.

In one embodiment, R₆ is hydrogen.

In one embodiment, R₇ is hydrogen, C₁₋₆alkoxy, or hydroxy.

In one embodiment, R₇ is hydrogen.

In one embodiment, R₈ is hydrogen.

In one embodiment, R₉ is hydrogen.

In one embodiment, R₉ is hydroxy.

In one embodiment, R₁₀ is hydrogen.

In one embodiment, R₁₀ is hydroxy.

In one embodiment, the compound of the invention is selected from thelist consisting of:

-   1-{[2-(1-ethyl-1H-pyrrolo[3,2-c]pyridin-2-yl)-1-methyl-1H-benzimidazol-5-yl]carbonyl}-3-piperidinamine;-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-c]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-5-methoxy-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-5-methoxy-1H-pyrrolo[2,3-c]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   2-(5-{[(3R)-3-amino-1-piperidinyl]carbonyl}-1-methyl-1H-benzimidazol-2-yl)-1-ethyl-1H-pyrrolo[2,3-b]pyridin-5-ol;-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(7-methoxy-1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone;-   (3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-7-(trifluoromethoxy)-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(7-methoxy-1-methyl-2-(1-neopentyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone;    ((R)-3-aminopiperidin-1-yl)(7-methoxy-1-methyl-2-(1-(2-methylbutyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone,    and;-   (R)-(3-aminopiperidin-1-yl)(7-methoxy-2-(1-(2-methoxy-2-methylpropyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;    and salts thereof.

In one embodiment, the compound of the invention is selected from thelist consisting of:

-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone    hydrochloride;-   (R)-(3-aminopiperidin-1-yl)(1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone    hydrochloride;-   (R)-(3-aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride;-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride;-   (R)-(3-aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride;-   (R)-(3-aminopiperidin-1-yl)(7-methoxy-1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride;-   ((3S,4R)-3-amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride;-   ((3S,4R)-3-amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride;-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride;-   (S)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride;-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-isobutyl-7-methoxy-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride;-   (R)-(3-aminopiperidin-1-yl)(2-(1-isobutyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride;-   ((2R,5S)-5-amino-2-methylpiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride, and;-   ((2R,5S)-5-amino-2-methylpiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride.

In one embodiment, the compound of the invention is selected from thelist consisting of:

-   ((3S,4R)-3-amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   ((3S,4R)-3-amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-5-yl)methanone;-   (S)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-isobutyl-7-methoxy-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(2-(1-isobutyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   ((2R,5S)-5-amino-2-methylpiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    and;-   ((2R,5S)-5-amino-2-methylpiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;    and salts thereof.

In one embodiment, the compound of the invention is selected from thelist consisting of:

-   (R)-(3-aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;-   (R)-(3-aminopiperidin-1-yl)(7-methoxy-1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone,    and;-   ((3S,4R)-3-amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;    and salts thereof.

In one embodiment, the compound of the invention is selected from thelist consisting of:

-   (R)-(3-aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride;-   (R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride;-   (R)-(3-aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride;-   (R)-(3-aminopiperidin-1-yl)(7-methoxy-1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride, and;-   ((3S,4R)-3-amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,    hydrochloride.

There is further provided a subset of compounds of formula (I), being offormula (I′)

wherein;

R₁, is hydrogen or C₁₋₆alkyl;

R₂ is hydrogen, C₁₋₆alkyl, or C₁₋₆alkoxy;

R₃ is hydrogen or C₁₋₆alkyl;

R₄, is hydrogen, C₁₋₆alkyl, perhalomethylC₁₋₆alkyl; or unsubstitutedC₃₋₆cycloalkylC₁₋₆alkyl;

A′ is C—R_(5′) or N;

B′ is C—R_(6′) or N;

D′ is C—R_(7′) or N;

with the proviso that at least one of A′, B′, and D′, is N;

R_(5′) is hydrogen or C₁₋₆alkyl;

R_(6′) is hydrogen or C₁₋₆alkyl;

R_(7′) is hydrogen, C₁₋₆alkyl, C₁₋₆alkoxy, or hydroxy;

R_(8′) is hydrogen or C₁₋₆alkyl, with the proviso that one of R_(4′) andR_(8′) is hydrogen;

and salts thereof.

It will be understood that references herein to compounds of formula (I)are equally-applicable to compounds of formula (I′), for example methodsof preparation, compositions, and methods of use.

TERMS AND DEFINITIONS

Compounds of Formula (I) and salts thereof are referred to hereinafteras ‘Compounds of the invention’.

‘Alkyl’ refers to a saturated hydrocarbon chain having the specifiednumber of carbon atoms. For example, C₁₋₆alkyl refers to an alkyl grouphaving from 1 to 6 carbon atoms, for example 1 to 2 carbon atoms. Alkylgroups may be straight or branched. Representative branched alkyl groupshave one, two, or three branches. ‘Alkyl’ includes methyl and ethyl.

‘Alkoxy’ refers to a saturated hydrocarbon chain having the specifiednumber of carbon atoms linked by a single bond to an oxygen atom. Forexample, C₁₋₆alkoxy refers to an alkoxy group having from 1 to 6 carbonatoms, for example 1 to 2 carbon atoms, for example 1 carbon atom.Alkoxy groups may be straight or branched. Representative branchedalkoxy groups have one, two, or three branches. ‘Alkoxy’ includesmethoxy.

‘Cycloalkyl’ refers to a saturated hydrocarbon ring having the specifiednumber of member atoms. For example, C₃₋₆cycloalkyl refers to acycloalkyl group having from 3 to 6 member atoms, for example 3 memberatoms. ‘Cycloalkyl’ includes cyclopropyl.

‘Enantiomeric excess’ (ee) is the excess of one enantiomer over theother expressed as a percentage. In a racemic modification, since bothenantiomers are present in equal amounts, the enantiomeric excess iszero (0% ee). However, if one enantiomer were enriched such that itconstitutes 95% of the product, then the enantiomeric excess would be90% ee (the amount of the enriched enantiomer, 95%, minus the amount ofthe other enantiomer, 5%).

‘Enantiomerically enriched’ refers to products whose enantiomeric excess(ee) is greater than zero. For example, ‘enantiomerically enriched’refers to products whose enantiomeric excess is greater than 50% ee,greater than 75% ee, and greater than 90% ee.

‘Enantiomerically pure’ refers to products whose enantiomeric excess is99% or greater.

‘Half-life’ (or ‘half-lives’) refers to the time required for half of aquantity of a substance to be converted to another chemically distinctspecies in vitro or in vivo.

‘Halo’ refers to a halogen radical, for example, fluoro, chloro, bromo,or iodo, for example bromo, chloro, or fluoro.

‘Perhalomethyl’ refers to a methyl group in which all of the hydrogenatoms have been replaced with a halogen radical. An example ofperhalomethyl is perfluoromethyl i.e. CF₃—.

‘Heterocyclic’ and ‘heterocyclyl’ refer to saturated or unsaturatedmonocyclic aliphatic rings containing 5, 6, or 7 ring members including1 or 2 heteroatoms or to saturated or unsaturated bicyclic aliphaticrings containing 6, 7, or 8 ring members including 1 or 2 heteroatoms.In certain embodiments, ‘heterocyclyl groups’ are saturated. In otherembodiments, ‘heterocyclyl’ groups are unsaturated. ‘Heterocyclyl’groups containing more than one heteroatom may contain differentheteroatoms. ‘Heterocyclyl’ groups may be substituted with one or moresubstituents as defined herein. ‘Heterocyclyl’ includes piperidinyl.

‘Heteroaryl’ refers to aromatic rings containing from 1 to 3 heteroatomsas member atoms in the ring. ‘Heteroaryl’ groups containing more thanone heteroatom may contain different heteroatoms. ‘Heteroaryl’ groupsmay be substituted with one or more substituents if so defined herein.The ‘heteroaryl’ rings have 5 or 6 member atoms. ‘Heteroaryl’ includespyrrolopyridinyl and benzimidazolyl.

‘Heteroatom’ refers to a nitrogen, sulfur, or oxygen atom, for example anitrogen atom.

‘Member atoms’ refers to the atom or atoms that form a chain or ring.Where more than one member atom is present in a chain and within a ring,each member atom is covalently bound to an adjacent member atom in thechain or ring. Atoms that make up a substituent group on a chain or ringare not member atoms in the chain or ring.

‘Substituted’ in reference to a group indicates that a hydrogen atomattached to a member atom within a group is replaced. It should beunderstood that the term ‘substituted’ includes the implicit provisionthat such substitution be in accordance with the permitted valence ofthe substituted atom and the substituent and that the substitutionresults in a stable compound (i.e. one that does not spontaneouslyundergo transformation such as rearrangement, cyclisation, orelimination). In certain embodiments, a single atom may be substitutedwith more than one substituent as long as such substitution is inaccordance with the permitted valence of the atom. Suitable substituentsare defined herein for each substituted or optionally substituted group.

‘Pharmaceutically acceptable’ refers to those compounds, materials,compositions, and dosage forms which are, within the scope of soundmedical judgment, suitable for use in contact with the tissues of humanbeings and animals without excessive toxicity, irritation, or otherproblem or complication, commensurate with a reasonable benefit/riskratio.

Throughout the description and the claims which follow, unless thecontext requires otherwise, the word ‘comprise’, and variations such as‘comprises’ and ‘comprising’, will be understood to imply the inclusionof a stated integer or step or group of integers but not to theexclusion of any other integer or step or group of integers or steps.

As used herein the symbols and conventions used in these processes,schemes and examples are consistent with those used in the contemporaryscientific literature, for example, the Journal of the American ChemicalSociety. Unless otherwise noted, all starting materials were obtainedfrom commercial suppliers and used without further purification.

Specifically, the following abbreviations may be used in the examplesand throughout the specification:

ABBREVIATIONS

-   AcOH Acetic acid-   BH₃-THF Borane tetrahydrofuran complex-   BOC/Boc tert-Butoxycarbonyl-   BOC₂O Di-tert-butyl dicarbonate-   nBuLi n-Butyllithium-   BuOH Butanol-   cHex Cyclohexane-   Cs₂CO₃ Caesium carbonate-   CV Column volumes-   DCM/CH₂Cl₂ Dichloromethane-   Dioxane 1,4-dioxane-   DIPEA N, N-diisopropylethylamine-   DMSO Dimethylsulfoxide-   DMF N,N-dimethylformamide-   Et₃N Triethylamine-   Ether Diethyl ether-   EtOAc Ethyl acetate-   GC Gas chromatography-   h. Hours-   HATU o-(7-Azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluronium    hexafluorophosphate-   HPLC High performance liquid chromatography-   K₂CO₃ Potassium carbonate-   KOH Potassium hydroxide-   LiCl Lithium chloride-   LiOH Lithium hydroxide-   LCMS or LC/MS Liquid chromatography-mass spectroscopy-   MDAP Mass directed automated preparative chromatography-   MeOH Methanol-   MeNH2 Methylamine-   min. Minutes-   Na₂SO₄ Sodium sulfate-   NaHCO₃ Sodium bicarbonate-   NH₄Cl Ammonium chloride-   NMP 1-Methyl-2-pyrrolidinone-   Pd/C Palladium on carbon-   PE Petroleum ether-   rb round-bottomed (flask)-   r.t/rt. Room temperature-   Rt Retention time-   SNAP Biotage™ flash chromatography cartridge-   SP4 Biotage™ flash purification system-   TFA Trifluoroacetic acid-   THF/thf Tetrahydrofuran-   TLC/tlc Thin layer chromatography-   TMEDA Tetramethylethylenediamine

Included within the scope of the ‘compounds of the invention’ are allsolvates (including hydrates), complexes, polymorphs, prodrugs,radiolabelled derivatives, and stereoisomers of the compounds of formula(I) and salts thereof.

The compounds of the invention may exist in solid or liquid form. In thesolid state, the compounds of the invention may exist in crystalline ornon-crystalline form, or as a mixture thereof. For compounds of theinvention that are in crystalline form, the skilled artisan willappreciate that pharmaceutically acceptable solvates may be formedwherein solvent molecules are incorporated into the crystalline latticeduring crystallization. Solvates may involve non-aqueous solvents suchas ethanol, iso-propyl alcohol, N,N-dimethylsulfoxide (DMSO), aceticacid, ethanolamine, and ethyl acetate, or they may involve water as thesolvent that is incorporated into the crystalline lattice. Solvateswherein water is the solvent that is incorporated into the crystallinelattice are typically referred to as ‘hydrates’. Hydrates includestoichiometric hydrates as well as compositions containing variableamounts of water. The invention includes all such solvates.

It will be further appreciated that certain compounds of the inventionthat exist in crystalline form, including the various solvates thereof,may exhibit polymorphism (i.e. the capacity to occur in differentcrystalline structures). These different crystalline forms are typicallyknown as ‘polymorphs’. The invention includes all such polymorphs.Polymorphs have the same chemical composition but differ in packing,geometrical arrangement, and other descriptive properties of thecrystalline solid state. Polymorphs, therefore, may have differentphysical properties such as shape, density, hardness, deformability,stability, and dissolution properties. Polymorphs typically exhibitdifferent melting points, IR spectra, and X-ray powder diffractionpatterns, which may be used for identification. It will be appreciatedthat different polymorphs may be produced, for example, by changing oradjusting the reaction conditions or reagents, used in making thecompound. For example, changes in temperature, pressure, or solvent mayresult in polymorphs. In addition, one polymorph may spontaneouslyconvert to another polymorph under certain conditions.

The invention also includes isotopically-labelled compounds, which areidentical to the compounds of formula (I) and salts thereof, but for thefact that one or more atoms are replaced by an atom having an atomicmass or mass number different from the atomic mass or mass number mostcommonly found in nature. Examples of isotopes that can be incorporatedinto the compounds of the invention include isotopes of hydrogen,carbon, nitrogen, oxygen and fluorine, such as ³H, ¹¹C, ¹⁴C and ¹⁸F.

The compounds according to formula (I) contain one or more asymmetriccentres (also referred to as a chiral centres) and may, therefore, existas individual enantiomers, diastereoisomers, or other stereoisomericforms, or as mixtures thereof. Chiral centres, such as chiral carbonatoms, may also be present in a substituent such as an alkyl group.Where the stereochemistry of a chiral centre present in formula (I), orin any chemical structure illustrated herein, is not specified, thestructure is intended to encompass any stereoisomer and all mixturesthereof. Thus, compounds according to formula (I) containing one or morechiral centres may be used as racemic modifications including racemicmixtures and racemates, enantiomerically-enriched mixtures, or asenantiomerically-pure individual stereoisomers. For example, thefragment (Z) of the compounds of formula (I) illustrated below:

contains a chiral centre at the junction of the amino-group with thering (marked with an asterisk (*)). The stereochemistry at this chiralcentre may be R, S, RS, or any mixture of R and S stereoisomers.

Individual stereoisomers of a compound according to formula (I) whichcontain one or more asymmetric centres may be resolved by methods knownto those skilled in the art. For example, such resolution may be carriedout (1) by formation of diastereoisomeric salts, complexes or otherderivatives; (2) by selective reaction with a stereoisomer-specificreagent, for example by enzymatic oxidation or reduction; or (3) bygas-liquid or liquid chromatography in a chiral environment, forexample, on a chiral support such as silica with a bound chiral ligandor in the presence of a chiral solvent. It will be appreciated thatwhere the desired stereoisomer is converted into another chemical entityby one of the separation procedures described above, a further step isrequired to liberate the desired form. Alternatively, specificstereoisomers may be synthesised by asymmetric synthesis using opticallyactive reagents, substrates, catalysts or solvents, or by converting oneenantiomer to the other by asymmetric transformation.

It is to be understood that the references herein to compounds offormula (I) and salts thereof covers the compounds of formula (I) asfree bases, or as salts thereof, for example as pharmaceuticallyacceptable salts thereof. Thus, in one embodiment, the invention isdirected to compounds of formula (I) as the free base. In anotherembodiment, the invention is directed to compounds of formula (I) andsalts thereof. In a further embodiment, the invention is directed tocompounds of formula (I) and pharmaceutically acceptable salts thereof.

It will be appreciated that pharmaceutically acceptable salts of thecompounds according to formula (I) may be prepared. Indeed, in certainembodiments of the invention, pharmaceutically acceptable salts of thecompounds according to formula (I) may be preferred over the respectivefree base because such salts impart greater stability or solubility tothe molecule thereby facilitating formulation into a dosage form.Accordingly, the invention is further directed to compounds of formula(I) and pharmaceutically acceptable salts thereof.

As used herein, the term ‘pharmaceutically acceptable salts’ refers tosalts that retain the desired biological activity of the subjectcompound and exhibit minimal undesired toxicological effects. Thesepharmaceutically acceptable salts may be prepared in situ during thefinal isolation and purification of the compound, or by separatelyreacting the purified compound in its free base form with a suitableacid.

Salts and solvates having non-pharmaceutically acceptable counter-ionsor associated solvents are within the scope of the present invention,for example, for use as intermediates in the preparation of othercompounds of formula (I) and their pharmaceutically acceptable salts.Thus one embodiment of the invention embraces compounds of formula (I)and salts thereof.

Compounds according to formula (I) contain a basic functional group andare therefore capable of forming pharmaceutically acceptable acidaddition salts by treatment with a suitable acid. Suitable acids includepharmaceutically acceptable inorganic acids and pharmaceuticallyacceptable organic acids. Representative pharmaceutically acceptableacid addition salts include hydrochloride, hydrobromide, nitrate,methylnitrate, sulfate, bisulfate, sulfamate, phosphate, acetate,hydroxyacetate, phenylacetate, propionate, butyrate, iso-butyrate,valerate, maleate, hydroxymaleate, acrylate, fumarate, malate, tartrate,citrate, salicylate, p-aminosalicyclate, glycollate, lactate,heptanoate, phthalate, oxalate, succinate, benzoate, o-acetoxybenzoate,chlorobenzoate, methylbenzoate, dinitrobenzoate, hydroxybenzoate,methoxybenzoate, naphthoate, hydroxynaphthoate, mandelate, tannate,formate, stearate, ascorbate, palmitate, oleate, pyruvate, pamoate,malonate, laurate, glutarate, glutamate, estolate, methanesulfonate(mesylate), ethanesulfonate (esylate), 2-hydroxyethanesulfonate,benzenesulfonate (besylate), p-aminobenzenesulfonate, p-toluenesulfonate(tosylate), and naphthalene-2-sulfonate.

Compound Preparation

The compounds of the invention may be made by a variety of methods,including standard chemistry. Any previously defined variable willcontinue to have the previously defined meaning unless otherwiseindicated. Illustrative general synthetic methods are set out in thefollowing schemes, and can be readily adapted to prepare other compoundsof the invention. Specific compounds of the invention are prepared inthe Examples section.

A compound of formula (I) may be prepared by deprotection of a compoundof formula (II). Accordingly, in a first aspect, there is provided aprocess for the preparation of a compound of formula (I) by deprotectionof a compound of formula (II):

wherein R₁, R₂, R₃, R₄, A, B, D, R₈, R₉, and R₁₀ are as hereinbeforedefined, and P is a suitable protecting group for amines, for exampletert-butoxycarbonyl (Boc), and thereafter, if required, preparing a saltof the compound so formed.

For example, to a solution of a compound of formula (II) in a suitablesolvent, for example dichloromethane, is added trifluoroacetic acid andthe reaction stirred at a suitable temperature, for example ambienttemperature, for a suitable period of time, for example 1-3 hours. Thereaction mixture is then concentrated under reduced pressure. The crudeproduct is then dissolved in a suitable solvent, for example methanol,and loaded onto an ion-exchange cartridge, for example a strongcation-exchange cartridge. The product is then eluted as the free basewith a suitable solvent, for example 2M ammonia in methanol and theeluant concentrated under reduced pressure to yield a compound offormula (I).

A compound of formula (II) may be prepared by condensation of a compoundof formula (III):

wherein R₁, R₂, R₃, R₉, R₁₀, and P are as hereinbefore defined, with acompound of formula (IV)

wherein R₄, A, B, D, and R₈ are as hereinbefore defined.

In a further aspect, there is provided a process for the preparation ofa compound of formula (II) by reaction of a compound of formula (III)with a compound of formula (IV).

For example, a compound of formula (IV) and a suitable peptide couplingreagent, for exampleo-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU), are dissolved in a suitable solvent, forexample N,N-dimethylformamide and stirred at a suitable temperature, forexample ambient temperature, for a suitable period of time, for example5-10 minutes. To this is added a solution of a compound of formula(III), and a suitable hindered base, for exampleN,N-di-iso-propylethylamine (DIPEA), in a suitable solvent, for exampleN,N-dimethylformamide, and the resulting mixture stirred under nitrogenat a suitable temperature, for example ambient temperature, for asuitable period of time, for example 3-5 hours. The reaction mixture isdiluted with water and partitioned with a suitable organic solvent, forexample ether. The organic layer is isolated then the aqueous layerre-extracted with ether. The combined organic layers are washed withwater then dried over sodium sulfate then passed through a hydrophobicfrit and concentrated under reduced pressure to give the crude amideintermediate. The solid is dried under reduced pressure for 12-24 hoursthen dissolved in a suitable solvent, for example toluene. A suitableorganic acid, for example acetic acid is added to the reaction mixtureand then the mixture is heated to reflux for a suitable period of time,for example which was refluxed for 4-6 hours. A suitable aqueous base,for example sodium bicarbonate solution is added to the reaction mixtureand the organic layer isolated. The aqueous layer is re-extracted with asuitable organic solvent, for example toluene, and the combined organiclayers concentrated under reduced pressure to give the crude product.The crude material may be purified by, for example, columnchromatography.

A compound of formula (II) may also be prepared by reaction of acompound of formula (XX):

wherein R₁, R₂, R₃, R₄, A, B, D, and R₈ are as hereinbefore defined,with a compound of formula (X) as hereinafter defined.

For example, to a solution of a compound of formula (XX) in a suitablesolvent, for example N,N-dimethylformamide (DMF) is added a suitablepeptide coupling reagent, for exampleo-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) followed by a suitable hindered base, forexample N,N-di-iso-propylethylamine (DIPEA), and the reaction stirred ata suitable temperature, for example ambient temperature, for a suitableperiod of time, for example 15-30 minutes. A compound of formula (X) isadded in a suitable solvent, for example DMF and the reaction stirred ata suitable temperature, for example ambient temperature, for a suitableperiod of time, for example 3-8 hours. Water and a suitable organicsolvent, for example diethyl ether are added and the layers separated.The aqueous layer is extracted with further organic solvent, for examplediethyl ether and the combined organic layers washed with water, dried,for example using anhydrous sodium sulfate, and concentrated underreduced pressure. The crude product may be purified for example bycolumn chromatography.

A compound of formula (III) may be prepared by reduction of a compoundof formula (VIII):

wherein R₁, R₂, R₃, R₉, R₁₀, and P are as hereinbefore defined.

For example, a compound of formula (VIII) is dissolved in a suitablesolvent, for example ethanol and added to a flushed hydrogenation flaskcontaining a suitable catalyst, for example palladium on carbon. Theresultant mixture is flushed with nitrogen/vacuum, then stirred under anatmosphere of hydrogen at a suitable temperature, for example ambienttemperature, for a suitable period of time, for example 24 hours. Thereaction mixture is then flushed from the hydrogen atmosphere withnitrogen/vacuum. To this solution, Celite is added and the mixturestirred for a suitable period of time, for example 2-5 minutes, thenfiltered under reduced pressure. The solution is concentrated underreduced pressure to give a crude product that may be purified by, forexample, chromatography.

A compound of formula (VIII) may be prepared by reaction of a compoundof formula (IX):

wherein R₁, R₂, and R₃ are as hereinbefore defined, with a compound offormula (X):

wherein R₉, R₁₀, and P are as hereinbefore defined.

For example, to a solution of a compound of formula (X), a compound offormula (IX) and a suitable peptide coupling reagent, for exampleo-(7-azabenzotriazol-1-yl)-N,N,N′,N′-tetramethyluroniumhexafluorophosphate (HATU) in a suitable solvent, for exampleN,N-dimethylformamide, is added a suitable hindered base, for exampleN,N-di-iso-propylethylamine (DIPEA) and the reaction stirred at asuitable temperature, for example ambient temperature, for a suitableperiod of time, for example 12-18 hours. Water and a suitable organicsolvent, for example diethyl ether, are added and the layers separated.The aqueous layer is extracted with further organic solvent, for examplediethyl ether, and the combined organic layers washed with water, dried,for example over anhydrous sodium sulfate, and concentrated in vacuo.The crude product may be purified using conventional techniques such aschromatography.

A compound of formula (IX) may be prepared by hydrolysis of a compoundof formula (XI):

wherein Rb is C₁₋₆alkyl and R₁, R₂, R₃ are as hereinbefore defined.

For example, a compound of formula (XI) is dissolved in a suitablesolvent, for example a 1:1 v/v ratio of tetrahydrofuran and water. Tothis solution is added a suitable base, for example lithium hydroxide,and the reaction stirred at a suitable temperature, for example ambienttemperature, for a suitable period of time, for example 12-18 hours. Thereaction mixture was cooled to a suitable temperature, for example 0° C.and acidified by the addition of a suitable aqueous mineral acid, forexample 5M HCl solution, until the pH reaches about 5. The slurry isfiltered and the product residue washed with distilled water and dried.

A compound of formula (XI) may be prepared by reaction of a compound offormula (XIV):

wherein Rb, R₁, and R₂, are as hereinbefore defined and L is a suitableleaving group, for example a halo group, for example chloro, with acompound of formula (XIII):R₃—NH₂  (XIII)wherein R₃ is as hereinbefore defined.

For example, a compound of formula (XIV) is dissolved in a suitablesolvent, for example N,N-dimethylformamide (DMF) and cooled to asuitable temperature, for example about 0° C. in an ice/water bath. Asolution of a compound of formula (XIII) in a suitable solvent, forexample tetrahydrofuran, is added dropwise with vigorous stirring andthe mixture flushed with nitrogen and heated to a suitable temperature,for example 70-90° C. for a suitable period of time, for example 3hours. The mixture is allowed to cool to a suitable temperature, forexample ambient temperature, over a suitable period of time, for example60-70 hours. The reaction mixture is diluted with water and filteredunder reduced pressure to give a compound of formula (XI).

A compound of formula (IV) wherein R₄ is other than hydrogen may beprepared by hydrolysis of a compound of formula (V):

wherein R_(a) is C₁₋₆alkyl, R₄, is other than hydrogen, and A, B, D, andR₈ are as hereinbefore defined.

For example, to a mixture of a compound of formula (V) in a suitablesolvent, for example a mixture of methanol, tetrahydrofuran (THF), andwater, is added a suitable base, for example lithium hydroxidemonohydrate, and the mixture stirred at a suitable temperature, forexample ambient temperature under an inert atmosphere, for example anatmosphere of nitrogen, for a suitable period of time, for example 1-2hours. The mixture is concentrated under reduced pressure, then treatedwith a suitable aqueous mineral acid, for example 2N HCl, and theproduct isolated by filtration.

A compound of formula (V) wherein R₄ is other than hydrogen may beprepared by alkylation of a compound of formula (IV) wherein R₄ ishydrogen i.e. a compound of formula (VI):

wherein A, B, D, and R₈ are as hereinbefore defined.

For example, a suitable organic solvent, for example dimethyl sulfoxide(DMSO) is added to a flask containing a suitable base, for examplepotassium hydroxide and the mixture stirred under an inert atmosphere,for example an atmosphere of nitrogen, for a suitable period of time,for example 8-12 minutes. A compound of formula (VI) and a suitablealkylating agent, for example bromoethane, is added and the mixturestirred at a suitable temperature, for example ambient temperature,under the inert atmosphere for a suitable period of time, for example18-24 hours. The reaction is quenched by the slow careful addition ofwater. A suitable organic solvent, for example diethyl ether, is addedand the reaction mixture separated into the organic and aqueous layers.The aqueous layer is further extracted with a suitable organic solvent,for example diethyl ether, and the combined organic layers dried, forexample by passing through a hydrophobic frit, and concentrated underreduced pressure to give a compound of formula (V) wherein R₄ is otherthan hydrogen.

A compound of formula (II) may also be prepared by condensation with acompound of formula (VII). Accordingly, in a further aspect, there isprovided a process for the preparation of a compound of formula (II) byreaction of a compound of formula (VII):

wherein R₄, A, B, D, and R₈, are as hereinbefore defined, with acompound of formula (VIII) as hereinbefore defined.

For example, to a solution of a compound of formula (VIII) and acompound of formula (VII) in a suitable solvent, for example ethanol, isadded portionwise a solution of a suitable reducing agent, for examplesodium dithionite in a suitable solvent, for example water. The mixtureis flushed with, for example nitrogen, then heated at a suitabletemperature, for example 1000C, for a suitable period of time, forexample 12-18 hours. The reaction mixture is concentrated under vacuumthen diluted with a suitable solvent, for example dichloromethane andwater added. The organic layer is collected and the aqueous layer washedwith further solvent, for example dichloromethane. The organic layersare combined, back washed with water, collected, dried with, for exampleanhydrous sodium sulfate, filtered through a hydrophobic frit andconcentrated under vacuum to yield the crude product. The crude productmay be purified by conventional means, for example chromatography.

A compound of formula (VII) wherein R₄ is other than hydrogen may beprepared by reaction of a compound of formula (VII) wherein R₄ ishydrogen by reaction with a compound of formula (XV):R₄-M  (XV)wherein R₄ is as hereinbefore defined and M is a suitable leaving group,for example a halo group, for example iodo, or an alkylsulfonyl group,for example trifluoromethanesulfonyl.

For example, to a suspension of compound of formula (VII) wherein R₄ ishydrogen and a suitable base, for example caesium carbonate, in asuitable organic medium, for example N,N-dimethylformamide stirred underan inert atmosphere, for example an atmosphere of nitrogen, at asuitable temperature, for example 20° C. is added a compound of formula(XV) dropwise over a suitable period of time, for example 0.5-1 minute.The reaction mixture is stirred at a suitable temperature, for exampleambient temperature. for a suitable period of time, for example 1 hour.The reaction mixture is quenched with water, partitioned between asuitable organic solvent, for example dichloromethane, and water. Theaqueous phase is extracted with a suitable organic solvent, for exampledichloromethane. The organic phase is washed with saturated brine, driedover, for example sodium sulfate, and evaporated in vacuo to give thecrude product. The crude product may be purified by conventional means,for example chromatography.

A compound of formula (VII) wherein R₄ is hydrogen may be prepared bydeprotection of a compound of formula (XVI):

wherein R_(c) is an aryl group, for example phenyl, and A, B, D, and R₈are as hereinbefore defined.

For example, to a solution of a suitable base, for example potassiumhydroxide, in a suitable organic solvent, for example methanol, stirredat a suitable temperature, for example ambient temperature, is added asolution of a compound of formula (XVI) in a suitable solvent, forexample methanol, dropwise over a suitable period of time, for example0.5-1 minute. The mixture is stirred at a suitable temperature, forexample ambient temperature, until the starting material is consumed.The reaction mixture is then diluted with water and then a suitableorganic solvent, for example dichloromethane is added. The pH isadjusted to 7 with a suitable mineral acid, for example concentratedhydrochloric acid, and extracted with further organic solvent, forexample dichloromethane. The organic phase is then washed, dried, andthe solvent removed to give a compound of formula (VII).

A compound of formula (XVI) may be prepared from a compound of formula(XVII)

wherein R_(c), A, B, D, and R₈ are as hereinbefore defined.

For example, to a solution of a suitable organic base, for examplediisopropylamine, in a suitable anhydrous organic solvent, for exampleanhydrous tetrahydrofuran, stirred under an inert atmosphere, forexample an atmosphere of nitrogen, at a suitable temperature, forexample −78° C. is added a suitable base, for example n-butyllithium,over a suitable period of time, for example 10-20 minutes. The reactionmixture is stirred at a suitable temperature, for example −78° C. for asuitable period of time, for example 20-40 minutes, then warmed to asuitable temperature, for example ambient temperature, and stirred for asuitable period of time, for example 45-90 minutes. To this solution oflithium diisopropylamide in a suitable anhydrous solvent, for exampleanhydrous tetrahydrofuran, stirred under an inert atmosphere, forexample an atmosphere of nitrogen, at a suitable temperature, forexample −30° C., is added a solution of a compound of formula (XVII) anda suitable base, for example tetramethylethylenediamine in a suitableorganic solvent, for example tetrahydrofuran, dropwise over a suitableperiod of time, for example 10-20 minutes. The reaction mixture isstirred at a suitable temperature, for example −30° C., for a suitableperiod of time, for example 2-3 hours, then a suitable organic solvent,for example N,N-dimethylformamide is added dropwise over a suitableperiod of time, for example 1 minute. The reaction mixture is stirred ata suitable temperature, for example −30° C. for a further suitableperiod of time, for example 1.5-3 hours. The reaction mixture isquenched with water and partitioned between a suitable organic solvent,for example dichloromethane, and water. The organic phase is washed,dried, and evaporated to give a crude compound of formula (XVI), whichmay be purified by conventional means, for example, recrystallisation.

A compound of formula (XVII) may be prepared by reaction of a compoundof formula (XVIII):

wherein A, B, D, and R₈ are as hereinbefore defined, with a compound offormula (XIX):R_(c)SO₂-Q  (XIX)wherein Rc is as hereinbefore defined and Q is a suitable leaving group,for example a halo group, for example chloro.

For example, to a solution of a compound of formula (XVIII) in asuitable organic solvent, for example tetrahydrofuran, is added asuitable base, for example sodium hydride portionwise over a suitableperiod of time, for example 5 minutes, under an inert atmosphere, forexample an atmosphere of nitrogen, at a suitable temperature, forexample 0° C. The reaction mixture is stirred at a suitable temperature,for example 0° C. for a suitable period of time, for example 30-45minutes, then a compound of formula (XIX) is added dropwise under aninert atmosphere, for example an atmosphere of nitrogen, at a suitabletemperature, for example 0° C., then stirred for a suitable period oftime, for example 1.5-3 hours at a suitable temperature, for exampleambient temperature, until the starting material had been completelyconsumed. The mixture is poured into water and extracted with a suitableorganic solvent, for example ethyl acetate. The organic phase is washed,dried, and evaporated to give a crude compound of formula (XVII), whichmay be purified by conventional means, for example, recrystallisation.

A compound of formula (XX) may be prepared by hydrolysis of a compoundof formula (XXI):

wherein R₁, R₂, R₃, R₄, A, B, D, and R₈ are as hereinbefore defined andR_(d) is an alkyl group, for example C₁₋₆alkyl,

For example, a compound of formula (XXI) is dissolved in a suitablesolvent, for example a mixture of a suitable organic solvent and water,for example a mixture of tetrahydrofuran (THF) and water, for example ina 1:1 ratio. To this is added a suitable base, for example lithiumhydroxide anhydrous and the reaction stirred at a suitable temperature,for example ambient temperature, for a suitable period of time, forexample 15-24 hours. The reaction mixture is then neutralised by theaddition of a suitable acid, for example 2M hydrochloric acid. Thesuspension is filtered and the residue washed with water and dried invacuo to afford a compound of formula (XX).

A compound of formula (XXI) may be prepared by reaction of a compound offormula (VII) as hereinbefore defined with a compound of formula (XI) ashereinbefore defined.

For example, a solution of a suitable reducing agent, for example sodiumhydrosulfite, in a suitable solvent, for example water is added to asuspension of a compound of formula (XI) and a compound of formula (VII)in a suitable medium, for example; ethanol. The reaction mixture isheated, for example in a microwave oven, to a suitable temperature, forexample, 90-110° C. for a suitable period of time, for example 3-6hours. The reaction mixture is diluted with a suitable solvent, forexample dichloromethane, dried, for example using anhydrous sodiumsulfate, filtered and concentrated under reduced pressure to afford thecrude product. The crude product may be purified by, for example, columnchromatography.

A compound of formula (X) wherein R₉ is hydroxy may be prepared byhydrogenolysis of a compound of formula (XXII):

wherein P and R₁₀ are as hereinbefore defined and P₁ is a protectinggroup, for example a carboxybenzyl group.

For example, a solution of a compound of formula (XXII) in a suitablesolvent, for example ethanol is added to a hydrogenation flaskcontaining a suitable catalyst, for example 10% palladium on carbon,under an inert atmosphere, for example an atmosphere of nitrogen. Theflask is then evacuated and back-filled with hydrogen. The system isclosed and the mixture allowed to stir under an atmosphere of hydrogenfor a suitable period of time, for example 12-18 hours. The reactionmixture was filtered and washed with a suitable solvent, for exampleethanol, followed by a further solvent wash with, for example ethylacetate. The combined filtrate is concentrated under reduced pressure toafford a compound of formula (X).

A compound of formula (XXII) wherein the —OH group and —NHP groups arecis with respect to each other may be prepared by hydrolysis of acompound of formula (XXIII):

wherein P and P₁, and R₁₀ are as hereinbefore defined and P₂ is asuitable protecting group, for example a benzoyl group.

For example, a solution of a suitable base, for example potassiumcarbonate in a suitable solvent, for example water is added to asolution of a compound of formula (XXIII) in a suitable solvent, forexample ethanol, and the mixture stirred at a suitable temperature, forexample 60-70° C. for 18-24 hours. The reaction mixture is concentratedunder reduced pressure, diluted with water, and extracted using asuitable solvent, for example dichloromethane. The organic extracts arecombined and dried using, for example anhydrous sodium sulfate, andconcentrated under reduced pressure to afford the crude compound offormula (XXII) wherein the —OH group and —NHP groups are cis withrespect to each other. The crude product may be purified by, forexample, column chromatography.

A compound of formula (XXIII) wherein the P₂O— and —NHP groups are ciswith respect to each other may be prepared from a compound of formula(XXII) where the HO— and —NHP groups are trans with respect to eachother via the Mitsunobu reaction.

For example, to a solution of triphenylphosphine in a suitable solvent,for example tetrahydrofuran is added di-iso-propyl azodicarboxylate andthe mixture was stirred at a suitable temperature, for example in anice-water bath for a suitable period of time, for example 10-15 minutesand then allowed to warm to ambient temperature. A compound of formula(XXII) where the HO— and —NHP groups are trans with respect to eachother in a suitable solvent, for example tetrahydrofuran, is addedfollowed by a suitable acid, for example benzoic acid. The reaction isstirred at a suitable temperature, for example ambient temperature, fora suitable period of time, for example 18-24 hours. The reaction mixtureis then concentrated, for example under reduced pressure. The crudeproduct is then purified susing, for example, column chromatography.

A compound of formula (XXII) wherein the —OH group and —NHP groups aretrans with respect to each other may be prepared by hydrolysis of acompound of formula (XXIV):

wherein P₁ and R₁₀ are as hereinbefore defined.

A solution of a compound of formula (XIV) in a suitable basic solventmixture, for example a mixture of aqueous ammonium hydroxide solutionand a suitable organic solvent, for example ethanol is stirred at asuitable temperature, for example 60-80° C. for a suitable period oftime, for example 4-6 hours. The reaction mixture is concentrated underreduced pressure, diluted with brine, and the organic layer extractedinto a suitable solvent, for example dichloromethane. The combinedorganic layers are dried using, for example anhydrous sodium sulfate,and concentrated under reduced pressure to give the intermediate primaryamine. The residue is diluted with a suitable solvent, for exampledichloromethane and a suitable base, for example triethylamine, and theprecursor to a suitable protecting group, for example di-tert-butyldicarbonate. The reaction is allowed to stir for a suitable period oftime, for example 1-3 hours, quenched with, for example, saturatedaqueous ammonium chloride solution, and the layers separated. Thecombined organic layers are dried using, for example, a hydrophobic fritand the solvent was removed under reduced pressure to yield a compoundof formula (XXII) wherein the —OH group and —NHP groups are trans withrespect to each other.

A compound of formula (XXIV) may be prepared by reaction of a suitableperacid, for example 3-chlorobenzoperoxoic acid, with a compound offormula (XXV):

wherein P₁ and R₁₀ are as hereinbefore defined.

For example, a suitable peracid, for example 3-chlorobenzoperoxoic acid,is added portionwise under an inert atmosphere, for example anatmosphere of nitrogen, to a stirred solution of a compound of formula(XXV) (available, for example, from Fluorochem, Hadfield, Derbyshire,UK) in a suitable anhydrous solvent, for example anhydrousdichloromethane, under cooling, for example using an ice bath. Theresulting mixture is allowed to reach ambient temperature and stirredfor a suitable period of time, for example 12-24 hours. Water is addedto the reaction mixture and the layers partitioned. The organic layer isadded to a stirred solution of a reducing agent for example an aqueoussolution of sodium metabisulfite to destroy excess peracid. The layersare separated and aqueous layer washed with a suitable solvent, forexample dichloromethane. The combined organic layers are then dried, forexample using anhydrous sodium sulfate, and concentrated under reducedpressure to afford the crude product, which may be purified bychromatography.

The preparations of compounds of formula (I) are summarised in thefollowing synthetic schemes:

Compounds of formulae (VI), (XIII), (X), (XIV), (XV), (XVIII), (XIX),and (XXV) are either known in the literature or are commerciallyavailable, for example from Sigma-Aldrich, UK, Apollo ScientificLimited, Alfa Aesar, Shanghai Haoyuan Chemexpress Co. Ltd, ActivateScientific GmbH, Lancaster Synthesis Ltd, Fluorochem, Hadfield,Derbyshire, UK, or may be prepared by analogy with known procedures, forexample those disclosed in standard reference texts of syntheticmethodology such as J. March, Advanced Organic Chemistry, 6th Edition(2007), WileyBlackwell, or Comprehensive Organic Synthesis (Trost B. M.and Fleming I., (Eds.), Pergamon Press, 1991), each incorporated hereinby reference as it relates to such procedures.

Examples of other protecting groups that may be employed in thesynthetic routes described herein and the means for their removal can befound in T. W. Greene ‘Protective Groups in Organic Synthesis’, 4thEdition, J. Wiley and Sons, 2006, incorporated herein by reference as itrelates to such procedures.

For any of the hereinbefore described reactions or processes,conventional methods of heating and cooling may be employed, for exampletemperature-regulated oil-baths or temperature-regulated hot-blocks, andice/salt baths or dry ice/acetone baths respectively. Conventionalmethods of isolation, for example extraction from or into aqueous ornon-aqueous solvents may be used. Conventional methods of drying organicsolvents, solutions, or extracts, such as shaking with anhydrousmagnesium sulfate, or anhydrous sodium sulfate, or passing through ahydrophobic frit, may be employed. Conventional methods of purification,for example crystallisation and chromatography, for example silicachromatography or reverse-phase chromatography, may be used as required.Crystallisation may be performed using conventional solvents such asethyl acetate, methanol, ethanol, or butanol, or aqueous mixturesthereof. It will be appreciated that specific reaction times andtemperatures may typically be determined by reaction-monitoringtechniques, for example thin-layer chromatography and LC-MS.

Where appropriate individual isomeric forms of the compounds of theinvention may be prepared as individual isomers using conventionalprocedures such as the fractional crystallisation of diastereoisomericderivatives or chiral high performance liquid chromatography (chiralHPLC).

The absolute stereochemistry of compounds may be determined usingconventional methods, such as X-ray crystallography or VCD (vibrationalcircular dichroism) analysis.

Methods of Use

The compounds of the invention are inhibitors of PAD4. Compounds whichinhibit PAD4 may be useful in the treatment of various disorders, forexample rheumatoid arthritis, vasculitis, systemic lupus erythematosus,ulcerative colitis, cancer, cystic fibrosis, asthma, cutaneous lupuserythematosis, and psoriasis.

The methods of treatment of the invention comprise administering a safeand effective amount of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, to a patient in need thereof. Individualembodiments of the invention include methods of treating any one of theabove-mentioned disorders by administering a safe and effective amountof a compound of formula (I), or a pharmaceutically acceptable saltthereof, to a patient in need thereof.

As used herein, ‘treat’ in reference to a disorder means: (1) toameliorate or prevent the disorder or one or more of the biologicalmanifestations of the disorder, (2) to interfere with (a). one or morepoints in the biological cascade that leads to or is responsible for thedisorder, or (b). one or more of the biological manifestations of thedisorder, (3) to alleviate one or more of the symptoms or effectsassociated with the disorder, or (4) to slow the progression of thedisorder or one or more of the biological manifestations of thedisorder.

As indicated above, ‘treatment’ of a disorder includes prevention of thedisorder. It will be appreciated that ‘prevention’ is not an absoluteterm. In medicine, ‘prevention’ is understood to refer to theprophylactic administration of a drug to substantially diminish thelikelihood or severity of a disorder or biological manifestationthereof, or to delay the onset of such disorder or biologicalmanifestation thereof.

As used herein, ‘safe and effective amount’ in reference to a compoundof formula (I), or a pharmaceutically acceptable salt thereof, or otherpharmaceutically-active agent means an amount of the compound sufficientto treat the patient's condition but low enough to avoid serious sideeffects (at a reasonable benefit/risk ratio) within the scope of soundmedical judgment. A safe and effective amount of a compound will varywith the particular compound chosen (for example, the potency, efficacy,and half-life of the compound will be considered); the route ofadministration chosen; the disorder being treated; the severity of thedisorder being treated; the age, size, weight, and physical condition ofthe patient being treated; the medical history of the patient to betreated; the duration of the treatment; the nature of concurrenttherapy; the desired therapeutic effect; and like factors, but cannevertheless be routinely determined by the skilled artisan.

As used herein, ‘patient’ refers to a human (including adults andchildren) or other animal. In one embodiment, ‘patient’ refers to ahuman.

The compounds of formula (I), or pharmaceutically acceptable saltsthereof, may be administered by any suitable route of administration,including both systemic administration and topical administration.Systemic administration includes oral administration, parenteraladministration, transdermal administration and rectal administration.Parenteral administration refers to routes of administration other thanenteral or transdermal, and is typically by injection or infusion.Parenteral administration includes intravenous, intramuscular, andsubcutaneous injection or infusion. Topical administration includesapplication to the skin as well as intraocular, otic, intravaginal,inhaled and intranasal administration. Inhalation refers toadministration into the patient's lungs whether inhaled through themouth or through the nasal passages. In one embodiment, the compounds offormula (I) or pharmaceutically acceptable salts thereof may beadministered orally. In another embodiment, the compounds of formula (I)or pharmaceutically acceptable salts thereof may be administeredtopically. In another embodiment, the compounds of formula (I) orpharmaceutically acceptable salts thereof may be administered byinhalation. In a further embodiment, the compounds of formula (I) orpharmaceutically acceptable salts thereof may be administeredintranasally.

The compounds of formula (I) or pharmaceutically acceptable saltsthereof may be administered once or according to a dosing regimenwherein a number of doses are administered at varying intervals of timefor a given period of time. For example, doses may be administered one,two, three, or four times per day. In one embodiment, a dose isadministered once per day. In a further embodiment, a dose isadministered twice per day. Doses may be administered until the desiredtherapeutic effect is achieved or indefinitely to maintain the desiredtherapeutic effect. Suitable dosing regimens for a compound of formula(I) or a pharmaceutically acceptable salt thereof depend on thepharmacokinetic properties of that compound, such as absorption,distribution, and half-life, which can be determined by the skilledartisan. In addition, suitable dosing regimens, including the durationsuch regimens are administered, for a compound of formula (I) or apharmaceutically acceptable salt thereof depend on the disorder beingtreated, the severity of the disorder being treated, the age andphysical condition of the patient being treated, the medical history ofthe patient to be treated, the nature of concurrent therapy, the desiredtherapeutic effect, and like factors within the knowledge and expertiseof the skilled artisan. It will be further understood by such skilledartisans that suitable dosing regimens may require adjustment given anindividual patient's response to the dosing regimen or over time asindividual patient needs change.

Typical daily dosages may vary depending upon the particular route ofadministration chosen. Typical daily dosages for oral administrationrange from 0.1 mg to 10 mg per kg of total body weight, for example from1 mg to 5 mg per kg of total body weight. For example, daily dosages fororal administration may be from 5 mg to 1 g per patient, such as 5 mg to500 mg per patient, or 5 mg to 250 mg.

Additionally, the compounds of formula (I) may be administered asprodrugs. As used herein, a ‘prodrug’ of a compound of formula (I) is afunctional derivative of the compound which, upon administration to apatient, eventually liberates the compound of formula (I) in vivo.Administration of a compound of formula (I) as a prodrug may enable theskilled artisan to do one or more of the following: (a) modify the onsetof the activity of the compound in vivo; (b) modify the duration ofaction of the compound in vivo; (c) modify the transportation ordistribution of the compound in vivo; (d) modify the solubility of thecompound in vivo; and (e) overcome a side effect or other difficultyencountered with the compound. Typical functional derivatives used toprepare prodrugs include modifications of the compound that arechemically or enzymatically cleavable in vivo. Such modifications, whichinclude the preparation of phosphates, amides, esters, thioesters,carbonates, and carbamates, are well known to those skilled in the art.

The invention thus provides a method of treating a disorder mediated byinappropriate PAD4 activity comprising administering a safe andeffective amount of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, to a patient in need thereof.

In one embodiment, the disorder mediated by inappropriate PAD4 activityis selected from the group consisting of rheumatoid arthritis,vasculitis, systemic lupus erythematosus, ulcerative colitis, cancer,cystic fibrosis, asthma, cutaneous lupus erythematosis, and psoriasis.In a further embodiment, the disorder mediated by inappropriate PAD4activity is rheumatoid arthritis. In a further embodiment, the disordermediated by inappropriate PAD4 activity is systemic lupus. In a furtherembodiment, the disorder mediated by inappropriate PAD4 activity isvasculitis. In a further embodiment, the disorder mediated byinappropriate PAD4 activity is cutaneous lupus erythematosis. In afurther embodiment, the disorder mediated by inappropriate PAD4 activityis psoriasis.

In one embodiment there is provided a method of treatment of rheumatoidarthritis, vasculitis, systemic lupus erythematosus, ulcerative colitis,cancer, cystic fibrosis, asthma, cutaneous lupus erythematosis, orpsoriasis, which method comprises administering to a human subject inneed thereof, a therapeutically effective amount of a compound offormula (I), or a pharmaceutically acceptable salt thereof.

In one embodiment there is provided a method of treatment of rheumatoidarthritis, which method comprises administering to a human subject inneed thereof, a therapeutically effective amount of a compound offormula (I), or a pharmaceutically acceptable salt thereof. In oneembodiment there is provided a method of treatment of systemic lupus,which method comprises administering to a human subject in need thereof,a therapeutically effective amount of a compound of formula (I), or apharmaceutically acceptable salt thereof. In one embodiment there isprovided a method of treatment of vasculitis, which method comprisesadministering to a human subject in need thereof, a therapeuticallyeffective amount of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof. In one embodiment there is provided a method oftreatment of cutaneous lupus erythematosis, which method comprisesadministering to a human subject in need thereof, a therapeuticallyeffective amount of a compound of formula (I), or a pharmaceuticallyacceptable salt thereof. In one embodiment there is provided a method oftreatment of psoriasis, which method comprises administering to a humansubject in need thereof, a therapeutically effective amount of acompound of formula (I), or a pharmaceutically acceptable salt thereof.

In one embodiment, the invention provides a compound of formula (I), ora pharmaceutically acceptable salt thereof, for use in therapy. Inanother embodiment, the invention provides a compound of formula (I), ora pharmaceutically acceptable salt thereof, for use in the treatment ofa disorder mediated by inappropriate PAD4 activity. In anotherembodiment, the invention provides a compound of formula (I), or apharmaceutically acceptable salt thereof, for use in the treatment ofrheumatoid arthritis, vasculitis, systemic lupus erythematosus,ulcerative colitis, cancer, cystic fibrosis, asthma, cutaneous lupuserythematosis, or psoriasis. In another embodiment, the inventionprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, for use in the treatment of rheumatoid arthritis. Inanother embodiment, the invention provides a compound of formula (I), ora pharmaceutically acceptable salt thereof, for use in the treatment ofsystemic lupus. In another embodiment, the invention provides a compoundof formula (I), or a pharmaceutically acceptable salt thereof, for usein the treatment of vasculitis. In another embodiment, the inventionprovides a compound of formula (I), or a pharmaceutically acceptablesalt thereof, for use in the treatment of cutaneous lupus erythematosis.In another embodiment, the invention provides a compound of formula (I),or a pharmaceutically acceptable salt thereof, for use in the treatmentof psoriasis. In another embodiment, the invention provides the use of acompound of formula (I), or a pharmaceutically acceptable salt thereof,in the manufacture of a medicament for use in the treatment of adisorder mediated by inappropriate PAD4 activity. In another embodiment,the invention provides the use of a compound of formula (I), or apharmaceutically acceptable salt thereof, in the manufacture of amedicament for use in the treatment of rheumatoid arthritis, vasculitis,systemic lupus erythematosus, ulcerative colitis, cancer, cysticfibrosis, asthma, cutaneous lupus erythematosis, or psoriasis. Inanother embodiment, the invention provides the use of a compound offormula (I), or a pharmaceutically acceptable salt thereof, in themanufacture of a medicament for use in the treatment of rheumatoidarthritis. In another embodiment, the invention provides the use of acompound of formula (I), or a pharmaceutically acceptable salt thereof,in the manufacture of a medicament for use in the treatment of systemiclupus. In another embodiment, the invention provides the use of acompound of formula (I), or a pharmaceutically acceptable salt thereof,in the manufacture of a medicament for use in the treatment ofvasculitis. In another embodiment, the invention provides the use of acompound of formula (I), or a pharmaceutically acceptable salt thereof,in the manufacture of a medicament for use in the treatment of cutaneouslupus erythematosis. In another embodiment, the invention provides theuse of a compound of formula (I), or a pharmaceutically acceptable saltthereof, in the manufacture of a medicament for use in the treatment ofpsoriasis. In a further embodiment, the invention provides apharmaceutical composition for the treatment or prophylaxis of adisorder mediated by inappropriate PAD4 activity comprising a compoundof formula (I) or a pharmaceutically acceptable salt thereof. In afurther embodiment, the invention provides a pharmaceutical compositionfor the treatment or prophylaxis of rheumatoid arthritis, vasculitis,systemic lupus erythematosus, ulcerative colitis, cancer, cysticfibrosis, asthma, cutaneous lupus erythematosis, or psoriasis,comprising a compound of formula (I) or a pharmaceutically acceptablesalt thereof. In a further embodiment, the invention provides apharmaceutical composition for the treatment or prophylaxis ofrheumatoid arthritis comprising a compound of formula (I) or apharmaceutically acceptable salt thereof. In a further embodiment, theinvention provides a pharmaceutical composition for the treatment orprophylaxis of systemic lupus comprising a compound of formula (I) or apharmaceutically acceptable salt thereof. In a further embodiment, theinvention provides a pharmaceutical composition for the treatment orprophylaxis of vasculitis comprising a compound of formula (I) or apharmaceutically acceptable salt thereof. In a further embodiment, theinvention provides a pharmaceutical composition for the treatment orprophylaxis of cutaneous lupus erythematosis comprising a compound offormula (I) or a pharmaceutically acceptable salt thereof. In a furtherembodiment, the invention provides a pharmaceutical composition for thetreatment or prophylaxis of psoriasis comprising a compound of formula(I) or a pharmaceutically acceptable salt thereof

Compositions

The compounds of formula (I) and pharmaceutically acceptable saltsthereof will normally, but not necessarily, be formulated intopharmaceutical compositions prior to administration to a patient.Accordingly, in another aspect there is provided a pharmaceuticalcomposition comprising a compound of formula (I), or a pharmaceuticallyacceptable salt thereof, and one or more pharmaceutically acceptableexcipients. In a further aspect the invention is directed topharmaceutical compositions for the treatment or prophylaxis of adisorder mediated by inappropriate PAD4 activity comprising a compoundof formula (I) or a pharmaceutically acceptable salt thereof.

The pharmaceutical compositions of the invention may be prepared andpackaged in bulk form wherein a safe and effective amount of a compoundof formula (I) or a pharmaceutically acceptable salt thereof can beextracted and then given to the patient such as with powders or syrups.Alternatively, the pharmaceutical compositions of the invention may beprepared and packaged in unit dosage form wherein each physicallydiscrete unit contains a compound of formula (I) or a pharmaceuticallyacceptable salt thereof. When prepared in unit dosage form, thepharmaceutical compositions of the invention typically may contain, forexample, from 0.25 mg to 1 g, or from 0.5 mg to 500 mg, or from 1 mg to100 mg, of a compound of formula (I) or a pharmaceutically acceptablesalt thereof.

The pharmaceutical compositions of the invention typically contain onecompound of formula (I) or a pharmaceutically acceptable salt thereof.

As used herein, ‘pharmaceutically acceptable excipient’ means apharmaceutically acceptable material, composition or vehicle involved ingiving form or consistency to the pharmaceutical composition. Eachexcipient must be compatible with the other ingredients of thepharmaceutical composition when commingled such that interactions whichwould substantially reduce the efficacy of the compound of formula (I)or a pharmaceutically acceptable salt thereof when administered to apatient and interactions which would result in pharmaceuticalcompositions that are not pharmaceutically acceptable are avoided. Inaddition, each excipient must of course be pharmaceutically acceptablee.g. of sufficiently high purity.

The compound of formula (I) or a pharmaceutically acceptable saltthereof and the pharmaceutically acceptable excipient or excipients willtypically be formulated into a dosage form adapted for administration tothe patient by the desired route of administration. For example, dosageforms include those adapted for (1) oral administration such as tablets,capsules, caplets, pills, troches, powders, syrups, elixers,suspensions, solutions, emulsions, sachets, and cachets; (2) parenteraladministration such as sterile solutions, suspensions, and powders forreconstitution; (3) transdermal administration such as transdermalpatches; (4) rectal administration such as suppositories; (5) inhalationsuch as aerosols, solutions, and dry powders; and (6) topicaladministration such as creams, ointments, lotions, solutions, pastes,sprays, foams, and gels.

Suitable pharmaceutically acceptable excipients will vary depending uponthe particular dosage form chosen. In addition, suitablepharmaceutically acceptable excipients may be chosen for a particularfunction that they may serve in the composition. For example, certainpharmaceutically acceptable excipients may be chosen for their abilityto facilitate the production of uniform dosage forms. Certainpharmaceutically acceptable excipients may be chosen for their abilityto facilitate the production of stable dosage forms. Certainpharmaceutically acceptable excipients may be chosen for their abilityto facilitate the carrying or transporting of the compound or compoundsof formula (I) or pharmaceutically acceptable salts thereof onceadministered to the patient from one organ, or portion of the body, toanother organ, or portion of the body. Certain pharmaceuticallyacceptable excipients may be chosen for their ability to enhance patientcompliance.

Suitable pharmaceutically-acceptable excipients include the followingtypes of excipients: Diluents, fillers, binders, disintegrants,lubricants, glidants, granulating agents, coating agents, wettingagents, solvents, co-solvents, suspending agents, emulsifiers,sweetners, flavouring agents, flavour-masking agents, colouring agents,anti-caking agents, humectants, chelating agents, plasticisers,viscosity increasing agents, antioxidants, preservatives, stabilisers,surfactants, and buffering agents. The skilled artisan will appreciatethat certain pharmaceutically-acceptable excipients may serve more thanone function and may serve alternative functions depending on how muchof the excipient is present in the formulation and what other excipientsare present in the formulation.

Skilled artisans possess the knowledge and skill in the art to enablethem to select suitable pharmaceutically-acceptable excipients inappropriate amounts for use in the invention. In addition, there are anumber of resources that are available to the skilled artisan whichdescribe pharmaceutically-acceptable excipients and may be useful inselecting suitable pharmaceutically-acceptable excipients. Examplesinclude Remington's Pharmaceutical Sciences (Mack Publishing Company),The Handbook of Pharmaceutical Additives (Gower Publishing Limited), andThe Handbook of Pharmaceutical Excipients (the American PharmaceuticalAssociation and the Pharmaceutical Press).

The pharmaceutical compositions of the invention are prepared usingtechniques and methods known to those skilled in the art. Some of themethods commonly used in the art are described in Remington'sPharmaceutical Sciences (Mack Publishing Company).

Accordingly, in another aspect the invention is directed to process forthe preparation of a pharmaceutical composition comprising a compound offormula (I) or a pharmaceutically acceptable salt thereof and one ormore pharmaceutically-acceptable excipients which comprises mixing theingredients. A pharmaceutical composition comprising a compound offormula (I) or a pharmaceutically acceptable salt thereof may beprepared by, for example, admixture at ambient temperature andatmospheric pressure.

In one embodiment, the compounds of formula (I) or pharmaceuticallyacceptable salts thereof will be formulated for oral administration. Inanother embodiment, the compounds of formula (I) or pharmaceuticallyacceptable salts thereof will be formulated for inhaled administration.In a further embodiment, the compounds of formula (I) orpharmaceutically acceptable salts thereof will be formulated forintranasal administration.

In one aspect, the invention is directed to a solid oral dosage formsuch as a tablet or capsule comprising a safe and effective amount of acompound of formula (I) or a pharmaceutically acceptable salt thereofand a diluent or filler. Suitable diluents and fillers include lactose,sucrose, dextrose, mannitol, sorbitol, starch (e.g. corn starch, potatostarch, and pre-gelatinized starch), cellulose and its derivatives (e.g.microcrystalline cellulose), calcium sulfate, and dibasic calciumphosphate. The oral solid dosage form may further comprise a binder.Suitable binders include starch (e.g. corn starch, potato starch, andpre-gelatinized starch), gelatin, acacia, sodium alginate, alginic acid,tragacanth, guar gum, povidone, and cellulose and its derivatives (e.g.microcrystalline cellulose). The oral solid dosage form may furthercomprise a disintegrant. Suitable disintegrants include crospovidone,sodium starch glycolate, croscarmelose, alginic acid, and sodiumcarboxymethyl cellulose. The oral solid dosage form may further comprisea lubricant. Suitable lubricants include stearic acid, magnesiumstearate, calcium stearate, and talc.

Where appropriate, dosage unit formulations for oral administration canbe microencapsulated. The composition can also be prepared to prolong orsustain the release as for example by coating or embedding particulatematerial in polymers, wax or the like.

The compounds of formula (I) or pharmaceutically acceptable saltsthereof may also be coupled with soluble polymers as targetable drugcarriers. Such polymers can include polyvinylpyrrolidone, pyrancopolymer, polyhydroxypropylmethacrylamide-phenol,polyhydroxyethylaspartamidephenol, or polyethyleneoxidepolylysinesubstituted with palmitoyl residues. Furthermore, the compounds offormula (I) or pharmaceutically acceptable salts thereof may be coupledto a class of biodegradable polymers useful in achieving controlledrelease of a drug, for example, polylactic acid, polepsiloncaprolactone, polyhydroxy butyric acid, polyorthoesters, polyacetals,polydihydropyrans, polycyanoacrylates and cross-linked or amphipathicblock copolymers of hydrogels.

In another aspect, the invention is directed to a liquid oral dosageform. Oral liquids such as solution, syrups and elixirs can be preparedin dosage unit form so that a given quantity contains a predeterminedamount of a compound of formula (I) or a pharmaceutically acceptablesalt thereof. Syrups can be prepared by dissolving the compound offormula (I) or a pharmaceutically acceptable salt thereof in a suitablyflavoured aqueous solution, while elixirs are prepared through the useof a non-toxic alcoholic vehicle. Suspensions can be formulated bydispersing the compound of formula (I) or a pharmaceutically acceptablesalt thereof in a non-toxic vehicle. Solubilisers and emulsifiers suchas ethoxylated isostearyl alcohols and polyoxy ethylene sorbitol ethers,preservatives, flavour additive such as peppermint oil or naturalsweeteners or saccharin or other artificial sweeteners, and the like canalso be added.

In another aspect, the invention is directed to a dosage form adaptedfor administration to a patient by inhalation, for example, as a drypowder, an aerosol, a suspension, or a solution composition.

Dry powder compositions for delivery to the lung by inhalation typicallycomprise a compound of formula (I) or a pharmaceutically acceptable saltthereof as a finely divided powder together with one or morepharmaceutically-acceptable excipients as finely divided powders.Pharmaceutically-acceptable excipients particularly suited for use indry powders are known to those skilled in the art and include lactose,starch, mannitol, and mono-, di-, and polysaccharides. The finelydivided powder may be prepared by, for example, micronisation andmilling. Generally, the size-reduced (eg micronised) compound can bedefined by a D₅₀ value of about 1 to about 10 microns (for example asmeasured using laser diffraction).

The dry powder may be administered to the patient via a reservoir drypowder inhaler (RDPI) having a reservoir suitable for storing multiple(un-metered doses) of medicament in dry powder form. RDPIs typicallyinclude a means for metering each medicament dose from the reservoir toa delivery position. For example, the metering means may comprise ametering cup, which is movable from a first position where the cup maybe filled with medicament from the reservoir to a second position wherethe metered medicament dose is made available to the patient forinhalation.

Alternatively, the dry powder may be presented in capsules (e.g. gelatinor plastic), cartridges, or blister packs for use in a multi-dose drypowder inhaler (MDPI). MDPIs are inhalers wherein the medicament iscomprised within a multi-dose pack containing (or otherwise carrying)multiple defined doses (or parts thereof) of medicament. When the drypowder is presented as a blister pack, it comprises multiple blistersfor containment of the medicament in dry powder form. The blisters aretypically arranged in regular fashion for ease of release of themedicament therefrom. For example, the blisters may be arranged in agenerally circular fashion on a disc-form blister pack, or the blistersmay be elongate in form, for example comprising a strip or a tape. Eachcapsule, cartridge, or blister may, for example, contain between 200μg-10 mg of the compound of formula (I) or a pharmaceutically acceptablesalt thereof.

Aerosols may be formed by suspending or dissolving a compound of formula(I) or a pharmaceutically acceptable salt thereof in a liquifiedpropellant. Suitable propellants include halocarbons, hydrocarbons, andother liquified gases. Representative propellants include:trichlorofluoromethane (propellant 11), dichlorofluoromethane(propellant 12), dichlorotetrafluoroethane (propellant 114),tetrafluoroethane (HFA-134a), 1,1-difluoroethane (HFA-152a),difluoromethane (HFA-32), pentafluoroethane (HFA-12), heptafluoropropane(HFA-227a), perfluoropropane, perfluorobutane, perfluoropentane, butane,isobutane, and pentane. Aerosols comprising a compound of formula (I) ora pharmaceutically acceptable salt thereof will typically beadministered to a patient via a metered dose inhaler (MDI). Such devicesare known to those skilled in the art.

The aerosol may contain additional pharmaceutically-acceptableexcipients typically used with MDIs such as surfactants, lubricants,cosolvents and other excipients to improve the physical stability of theformulation, to improve valve performance, to improve solubility, or toimprove taste.

There is thus provided as a further aspect of the invention apharmaceutical aerosol formulation comprising a compound of formula (I)or a pharmaceutically acceptable salt thereof and a fluorocarbon orhydrogen-containing chlorofluorocarbon as propellant, optionally incombination with a surfactant and/or a cosolvent.

According to another aspect of the invention, there is provided apharmaceutical aerosol formulation wherein the propellant is selectedfrom 1,1,1,2-tetrafluoroethane, 1,1,1,2,3,3,3-heptafluoro-n-propane andmixtures thereof.

The formulations of the invention may be buffered by the addition ofsuitable buffering agents.

Capsules and cartridges for use in an inhaler or insufflator, of forexample gelatine, may be formulated containing a powder mix forinhalation of a compound of formula (I) or a pharmaceutically acceptablesalt thereof and a suitable powder base such as lactose or starch. Eachcapsule or cartridge may generally contain from 200 μg to 10 mg of thecompound of formula (I) or pharmaceutically acceptable salt thereof.Alternatively, the compound of formula (I) or pharmaceuticallyacceptable salt thereof may be presented without excipients such aslactose.

The proportion of the active compound of formula (I) or pharmaceuticallyacceptable salt thereof in the local compositions according to theinvention depends on the precise type of formulation to be prepared butwill generally be within the range of from 0.01 to 10% by weight.Generally, for most types of preparations, the proportion used will bewithin the range of from 0.05 to 1%, for example from 0.1 to 0.5%.

Aerosol formulations are preferably arranged so that each metered doseor ‘puff’ of aerosol contains from 20 μg to 10 mg, preferably from 20 μgto 5 mg, more preferably from about 20 μg to 0.5 mg of a compound offormula (I). Administration may be once daily or several times daily,for example 2, 3, 4 or 8 times, giving for example 1, 2 or 3 doses eachtime. The overall daily dose with an aerosol will be within the rangefrom 100 μg to 10 mg, for example from 200 μg to 5 mg. The overall dailydose and the metered dose delivered by capsules and cartridges in aninhaler or insufflator will generally be double that delivered withaerosol formulations.

In the case of suspension aerosol formulations, the particle size of theparticulate (e.g., micronised) drug should be such as to permitinhalation of substantially all the drug into the lungs uponadministration of the aerosol formulation and will thus be less than 100microns, desirably less than 20 microns, and in particular in the rangeof from 1 to 10 microns, such as from 1 to 5 microns, more preferablyfrom 2 to 3 microns.

The formulations of the invention may be prepared by dispersal ordissolution of the medicament and a compound of formula (I) or apharmaceutically acceptable salt thereof in the selected propellant inan appropriate container, for example, with the aid of sonication or ahigh-shear mixer. The process is desirably carried out under controlledhumidity conditions.

The chemical and physical stability and the pharmaceutical acceptabilityof the aerosol formulations according to the invention may be determinedby techniques well known to those skilled in the art. Thus, for example,the chemical stability of the components may be determined by HPLCassay, for example, after prolonged storage of the product. Physicalstability data may be gained from other conventional analyticaltechniques such as, for example, by leak testing, by valve deliveryassay (average shot weights per actuation), by dose reproducibilityassay (active ingredient per actuation) and spray distribution analysis.

The stability of the suspension aerosol formulations according to theinvention may be measured by conventional techniques, for example, bymeasuring flocculation size distribution using a back light scatteringinstrument or by measuring particle size distribution by cascadeimpaction or by the ‘twin impinger’ analytical process. As used hereinreference to the ‘twin impinger’ assay means ‘Determination of thedeposition of the emitted dose in pressurised inhalations usingapparatus A’ as defined in British Pharmacopaeia 1988, pages A204-207,Appendix XVII C. Such techniques enable the ‘respirable fraction’ of theaerosol formulations to be calculated. One method used to calculate the‘respirable fraction’ is by reference to ‘fine particle fraction’ whichis the amount of active ingredient collected in the lower impingementchamber per actuation expressed as a percentage of the total amount ofactive ingredient delivered per actuation using the twin impinger methoddescribed above.

The term ‘metered dose inhaler’ or MDI means a unit comprising a can, asecured cap covering the can and a formulation metering valve situatedin the cap. MDI system includes a suitable channeling device. Suitablechanneling devices comprise for example, a valve actuator and acylindrical or cone-like passage through which medicament may bedelivered from the filled canister via the metering valve to the nose ormouth of a patient such as a mouthpiece actuator.

MDI canisters generally comprise a container capable of withstanding thevapour pressure of the propellant used such as a plastic orplastic-coated glass bottle or preferably a metal can, for example,aluminium or an alloy thereof which may optionally be anodised,lacquer-coated and/or plastic-coated (for example incorporated herein byreference WO 96/32099 wherein part or all of the internal surfaces arecoated with one or more fluorocarbon polymers optionally in combinationwith one or more non-fluorocarbon polymers), which container is closedwith a metering valve. The cap may be secured onto the can viaultrasonic welding, screw fitting or crimping. MDIs taught herein may beprepared by methods of the art (e.g. see Byron, above and WO 96/32099).Preferably the canister is fitted with a cap assembly, wherein adrug-metering valve is situated in the cap, and said cap is crimped inplace.

In one embodiment of the invention the metallic internal surface of thecan is coated with a fluoropolymer, more preferably blended with anon-fluoropolymer. In another embodiment of the invention the metallicinternal surface of the can is coated with a polymer blend ofpolytetrafluoroethylene (PTFE) and polyethersulfone (PES). In a furtherembodiment of the invention the whole of the metallic internal surfaceof the can is coated with a polymer blend of polytetrafluoroethylene(PTFE) and polyethersulfone (PES).

The metering valves are designed to deliver a metered amount of theformulation per actuation and incorporate a gasket to prevent leakage ofpropellant through the valve. The gasket may comprise any suitableelastomeric material such as, for example, low density polyethylene,chlorobutyl, bromobutyl, EPDM, black and white butadiene-acrylonitrilerubbers, butyl rubber and neoprene. Suitable valves are commerciallyavailable from manufacturers well known in the aerosol industry, forexample, from Valois, France (e.g. DF10, DF30, DF60), Bespak plc, UK(e.g. BK300, BK357) and 3M-Neotechnic Ltd, UK (e.g. Spraymiser™).

In various embodiments, the MDIs may also be used in conjunction withother structures such as, without limitation, overwrap packages forstoring and containing the MDIs, including those described in U.S. Pat.Nos. 6,119,853; 6,179,118; 6,315,112; 6,352,152; 6,390,291; and6,679,374, as well as dose counter units such as, but not limited to,those described in U.S. Pat. Nos. 6,360,739 and 6,431,168.

Conventional bulk manufacturing methods and machinery well known tothose skilled in the art of pharmaceutical aerosol manufacture may beemployed for the preparation of large-scale batches for the commercialproduction of filled canisters. Thus, for example, in one bulkmanufacturing method for preparing suspension aerosol formulations ametering valve is crimped onto an aluminium can to form an emptycanister. The particulate medicament is added to a charge vessel andliquefied propellant together with the optional excipients is pressurefilled through the charge vessel into a manufacturing vessel. The drugsuspension is mixed before recirculation to a filling machine and analiquot of the drug suspension is then filled through the metering valveinto the canister. In one example bulk manufacturing method forpreparing solution aerosol formulations a metering valve is crimped ontoan aluminium can to form an empty canister. The liquefied propellanttogether with the optional excipients and the dissolved medicament ispressure filled through the charge vessel into a manufacturing vessel.

In an alternative process, an aliquot of the liquefied formulation isadded to an open canister under conditions which are sufficiently coldto ensure the formulation does not vaporise, and then a metering valvecrimped onto the canister.

Typically, in batches prepared for pharmaceutical use, each filledcanister is check-weighed, coded with a batch number and packed into atray for storage before release testing.

Suspensions and solutions comprising a compound of formula (I) or apharmaceutically acceptable salt thereof may also be administered to apatient via a nebuliser. The solvent or suspension agent utilized fornebulization may be any pharmaceutically-acceptable liquid such aswater, aqueous saline, alcohols or glycols, e.g., ethanol,isopropylalcohol, glycerol, propylene glycol, polyethylene glycol, etc.or mixtures thereof. Saline solutions utilize salts which display littleor no pharmacological activity after administration. Both organic salts,such as alkali metal or ammonium halogen salts, e.g., sodium chloride,potassium chloride or organic salts, such as potassium, sodium andammonium salts or organic acids, e.g., ascorbic acid, citric acid,acetic acid, tartaric acid, etc. may be used for this purpose.

Other pharmaceutically-acceptable excipients may be added to thesuspension or solution. The compound of formula (I) or pharmaceuticallyacceptable salt thereof may be stabilized by the addition of aninorganic acid, e.g., hydrochloric acid, nitric acid, sulfuric acidand/or phosphoric acid; an organic acid, e.g., ascorbic acid, citricacid, acetic acid, and tartaric acid, etc., a complexing agent such asEDTA or citric acid and salts thereof; or an antioxidant such asantioxidant such as vitamin E or ascorbic acid. These may be used aloneor together to stabilize the compound of formula (I) or pharmaceuticallyacceptable salt thereof. Preservatives may be added such as benzalkoniumchloride or benzoic acid and salts thereof. Surfactant may be addedparticularly to improve the physical stability of suspensions. Theseinclude lecithin, disodium dioctylsulfosuccinate, oleic acid andsorbitan esters.

In a further aspect, the invention is directed to a dosage form adaptedfor intranasal administration.

Formulations for administration to the nose may include pressurisedaerosol formulations and aqueous formulations administered to the noseby pressurised pump. Formulations which are non-pressurised and adaptedto be administered topically to the nasal cavity are of particularinterest. Suitable formulations contain water as the diluent or carrierfor this purpose. Aqueous formulations for administration to the lung ornose may be provided with conventional excipients such as bufferingagents, tonicity modifying agents and the like. Aqueous formulations mayalso be administered to the nose by nebulisation.

The compounds of formula (I) or pharmaceutically acceptable saltsthereof may be formulated as a fluid formulation for delivery from afluid dispenser, for example a fluid dispenser having a dispensingnozzle or dispensing orifice through which a metered dose of the fluidformulation is dispensed upon the application of a user-applied force toa pump mechanism of the fluid dispenser. Such fluid dispensers aregenerally provided with a reservoir of multiple metered doses of thefluid formulation, the doses being dispensable upon sequential pumpactuations. The dispensing nozzle or orifice may be configured forinsertion into the nostrils of the user for spray dispensing of thefluid formulation into the nasal cavity. A fluid dispenser of theaforementioned type is described and illustrated in WO 05/044354, theentire content of which is hereby incorporated herein by reference. Thedispenser has a housing which houses a fluid discharge device having acompression pump mounted on a container for containing a fluidformulation. The housing has at least one finger-operable side leverwhich is movable inwardly with respect to the housing to cam thecontainer upwardly in the housing to cause the pump to compress and pumpa metered dose of the formulation out of a pump stem through a nasalnozzle of the housing. In one embodiment, the fluid dispenser is of thegeneral type illustrated in FIGS. 30-40 of WO 05/044354.

Pharmaceutical compositions adapted for intranasal administrationwherein the carrier is a solid include a coarse powder having a particlesize for example in the range 20 to 500 microns which is administered byrapid inhalation through the nasal passage from a container of thepowder held close up to the nose. Suitable compositions wherein thecarrier is a liquid, for administration as a nasal spray or as nasaldrops, include aqueous or oil solutions of the compound of formula (I)or a pharmaceutically acceptable salt thereof.

Pharmaceutical compositions adapted for transdermal administration maybe presented as discrete patches intended to remain in intimate contactwith the epidermis of the patient for a prolonged period of time. Forexample, the active ingredient may be delivered from the patch byiontophoresis as generally described in Pharmaceutical Research, 3(6),318 (1986).

Pharmaceutical compositions adapted for topical administration may beformulated as ointments, creams, suspensions, lotions, powders,solutions, pastes, gels, sprays, aerosols or oils.

Ointments, creams and gels, may, for example, be formulated with anaqueous or oily base with the addition of suitable thickening and/orgelling agent and/or solvents. Such bases may thus, for example, includewater and/or an oil such as liquid paraffin or a vegetable oil such asarachis oil or castor oil, or a solvent such as polyethylene glycol.Thickening agents and gelling agents which may be used according to thenature of the base include soft paraffin, aluminium stearate,cetostearyl alcohol, polyethylene glycols, woolfat, beeswax,carboxypolymethylene and cellulose derivatives, and/or glycerylmonostearate and/or non-ionic emulsifying agents.

Lotions may be formulated with an aqueous or oily base and will ingeneral also contain one or more emulsifying agents, stabilising agents,dispersing agents, suspending agents or thickening agents.

Powders for external application may be formed with the aid of anysuitable powder base, for example, talc, lactose or starch. Drops may beformulated with an aqueous or non-aqueous base also comprising one ormore dispersing agents, solubilising agents, suspending agents orpreservatives.

Topical preparations may be administered by one or more applications perday to the affected area. Over skin areas, occlusive dressings mayadvantageously be used. Continuous or prolonged delivery may be achievedby an adhesive reservoir system.

For treatments of the eye or other external tissues, for example mouthand skin, the compositions may be applied as a topical ointment orcream. When formulated in an ointment, the compound of formula (I) or apharmaceutically acceptable salt thereof may be employed with either aparaffinic or a water-miscible ointment base. Alternatively, thecompound of formula (I) or pharmaceutically acceptable salt thereof maybe formulated in a cream with an oil-in-water cream base or awater-in-oil base.

Pharmaceutical compositions adapted for parenteral administrationinclude aqueous and non-aqueous sterile injection solutions which maycontain anti-oxidants, buffers, bacteriostats and solutes which renderthe formulation isotonic with the blood of the intended recipient; andaqueous and non-aqueous sterile suspensions which may include suspendingagents and thickening agents. The compositions may be presented inunit-dose or multi-dose containers, for example sealed ampoules andvials, and may be stored in a freeze-dried (lyophilized) conditionrequiring only the addition of the sterile liquid carrier, for examplewater for injections, immediately prior to use. Extemporaneous injectionsolutions and suspensions may be prepared from sterile powders, granulesand tablets.

The invention will now be illustrated by way of the followingnon-limiting examples.

General Methods

Unless stated otherwise, starting materials were commercially available.All solvents and commercial reagents were of laboratory grade and wereused as received.

Where diastereoisomers are represented and only the relativestereochemistry is referred to, the bold or hashed solid bond symbols (

/

) are used. Where the absolute stereochemistry is known and the compoundis a single enantiomer, the bold or hashed wedges symbols (

/

) are used as appropriate.

Analytical Methods

Method A

LCMS was conducted on an Acquity UPLC BEH C₁₈ column (50 mm×2.1 mm i.d.1.7 μm packing diameter) at 40 degrees centigrade, eluting with 10 mMammonium bicarbonate in water adjusted to pH 10 with ammonia solution(Solvent A) and acetonitrile (Solvent B) using the following elutiongradient: 0-1.5 min: 1-97% B, 1.5-1.9 min: 97% B, 1.9-2.0 min: 100% B ata flow rate of 1 ml/min. The UV detection was a summed signal fromwavelength of 210 nm to 350 nm. The mass spectra were recorded on aWaters ZQ Mass Spectrometer using Alternate-scan Positive and NegativeElectrospray. Ionisation data were rounded to the nearest integer.

Method B

LCMS was conducted on an Acquity UPLC BEH C₁₈ column (50 mm×2.1 mm i.d.1.7 μm packing diameter) at 40 degrees centigrade, eluting with 0.1% v/vsolution of formic acid in water (Solvent A) and 0.1% v/v solution offormic acid in acetonitrile (Solvent B) using the following elutiongradient: 0-1.5 min: 3-100% B, 1.5-1.9 min: 100% B, 1.9-2.0 min: 3% B ata flow rate of 1 ml/min. The UV detection was a summed signal fromwavelength of 210 nm to 350 nm. The mass spectra were recorded on aWaters ZQ Mass Spectrometer using Alternate-scan Positive and NegativeElectrospray. Ionisation data were rounded to the nearest integer.

Method C

LCMS was conducted on an Acquity UPLC BEH C₁₈ column (50 mm×2.1 mm i.d.1.7 μm packing diameter) at 40 degrees centigrade, eluting with 0.1% v/vsolution of trifluoroacetic acid in water (Solvent A) and 0.1% v/vsolution of trifluoroacetic acid in acetonitrile (Solvent B) using thefollowing elution gradient: 0-1.5 min: 3-100% B, 1.5-1.9 min: 100% B,1.9-2.0 min: 3% B at a flow rate of 1 ml/min. The UV detection was asummed signal from wavelength of 210 nm to 350 nm. The mass spectra wererecorded on a Waters ZQ Mass Spectrometer using Alternate-scan Positiveand Negative Electrospray. Ionisation data were rounded to the nearestinteger.

Method D

LCMS was conducted on a HALO C₁₈ column (50 mm×4.6 mm i.d. 2.7 μmpacking diameter) at 40 degrees centigrade, eluting with 0.1% v/vsolution of formic acid in water (Solvent A) and 0.1% v/v solution offormic acid in acetonitrile (Solvent B) using the following elutiongradient: 0-1.8 min: 5% B, 1.8-2.01 min: 100% B, 2.01-2.8 min: 5% B at aflow rate of 1.5 ml/min. The UV detection was a summed signal atwavelength: 214 nm and 254 nm. MS: Ion Source: ESI; Detector Voltage:1.4 KV; Heat Block temp.: 250° C.; CDL temp.: 250° C.; Nebuliser GasFlow: 1.5 mL/min.

Method E

LCMS was conducted on a HALO C₁₈ column (50 mm×4.6 mm i.d. 2.7 μmpacking diameter) at 40 degrees centigrade, eluting with 0.1% v/vsolution of formic acid in water (Solvent A) and 0.1% v/v solution offormic acid in acetonitrile (Solvent B) using the following elutiongradient: 0-1 min: 5% B, 1-2.01 min: 95% B, 2.01-2.5 min: 5% B at a flowrate of 1.8 ml/min. The UV detection was a summed signal at wavelength:214 nm and 254 nm. MS: Ion Source: ESI; Drying Gas Flow: 10 L/min;Nebuliser Pressure: 45 psi; Drying Gas Temperature: 330° C.; CapillaryVoltage: 4000V.

General GC Method

GCMS was conducted on an Agilent 6890/5973 GCMS equipment with anAgilent capillary column HP-5 (0.25 um×30 m, i.d. 0.25 mm). The initialtemperature is 50° C. The equilibration time is 0.50 min. The initialtime is 1.00 min. The temperature then increase to 180° C. with a rateof 10°/min, then rise to 240° C. with a rate of 20° C./min, then hold240° C. for 5.00 min. The injection mode is splitless. The gas flow is1.00 ml/min and the total flow is 23.2 ml/min. The average velocity is36 cm/sec. The acquisition mode is scan. The ionization method is 70 eVEI (Electronic Ionization).

¹H NMR spectra were recorded using a Bruker DPX 400 MHz or AV 600 MHzspectrometer, referenced to tetramethylsilane.

Silica chromatography techniques include either automated (Flashmaster,Biotage SP4) techniques or manual chromatography on pre-packedcartridges (SPE) or manually-packed flash columns.

When the name of a commercial supplier is given after the name of acompound or a reagent, for instance “compound X (Aldrich)” or “compoundX/Aldrich”, this means that compound X is obtainable from a commercialsupplier, such as the commercial supplier named.

Similarly, when a literature or a patent reference is given after thename of a compound, for instance ‘compound Y (EP 0 123 456)’, this meansthat the preparation of the compound is described in the namedreference.

The names of the intermediates and examples have been obtained using thecompound naming programme within ChemBioDraw Ultra v12, or alternativelyusing “ACD Name Pro 6.02”.

General MDAP Purification Methods

Listed below are examples of mass-directed autopreparativechromatography (MDAP) methods that have been used or may be used incompound purification.

MDAP (Method A). The HPLC analysis is conducted on an XBridge C₁₈ column(100 mm×30 mm i.d. 5 μm packing diameter) at ambient temperature,eluting with 10 mM ammonium bicarbonate in water adjusted to pH 10 withammonia solution (Solvent A) and acetonitrile (Solvent B) using thefollowing elution gradient:

Time (min) Flow Rate (ml/min) % A % B 0 40 85 15 1 40 85 15 10 40 45 5511 40 1 99 15 40 1 99

The UV detection is an averaged signal from wavelength of 210 nm to 350nm. The mass spectra are recorded on a Waters ZQ Mass Spectrometer usingAlternate-scan Positive and Negative Electrospray. Ionisation data arerounded to the nearest integer.

MDAP (Method B). The HPLC analysis is conducted on an XBridge C₁₈ column(100 mm×30 mm i.d. 5 μm packing diameter) at ambient temperature,eluting with 10 mM ammonium bicarbonate in water adjusted to pH 10 withammonia solution (Solvent A) and acetonitrile (Solvent B) using thefollowing elution gradient:

Time (min) Flow Rate (ml/min) % A % B 0 40 85 15 1 40 85 15 20 40 45 5521 40 1 99 25 40 1 99

The UV detection is an averaged signal from wavelength of 210 nm to 350nm. The mass spectra are recorded on a Waters ZQ Mass Spectrometer usingAlternate-scan Positive and Negative Electrospray. Ionisation data arerounded to the nearest integer.

MDAP (Method C). The HPLC analysis is conducted on a Sunfire C₁₈ column(150 mm×30 mm i.d. 5 μm packing diameter) at ambient temperature,eluting with 0.1% v/v solution of trifluoroacetic acid in water (SolventA) and 0.1% v/v solution of trifluoroacetic acid in acetonitrile(Solvent B) using the following elution gradient:

Time (min) Flow Rate (ml/min) % A % B 0 40 100 0 3 40 100 0 3.5 30 100 024.5 30 70 30 25 30 1 99 32 30 1 99

The UV detection is an averaged signal from wavelength of 210 nm to 350nm. The mass spectra are recorded on a Waters ZQ Mass Spectrometer usingAlternate-scan Positive and Negative Electrospray. Ionisation data arerounded to the nearest integer.

MDAP (Method D). The HPLC analysis is conducted on a Sunfire C₁₈ column(150 mm×30 mm i.d. 5 μm packing diameter) at ambient temperature,eluting with 10 mM ammonium bicarbonate in water adjusted to pH 10 withammonia solution (Solvent A) and acetonitrile (Solvent B) using thefollowing elution gradient:

Time (min) Flow Rate (ml/min) % A % B 0 40 100 0 3 40 100 0 3.5 30 100 024.5 30 70 30 25 30 1 99 32 30 1 99

The UV detection is an averaged signal from wavelength of 210 nm to 350nm. The mass spectra are recorded on a Waters ZQ Mass Spectrometer usingAlternate-scan Positive and Negative Electrospray. Ionisation data arerounded to the nearest integer.

MDAP (Method E). The HPLC analysis was conducted on an XBridge C18column (100 mm×30 mm i.d. 5 μm packing diameter) at ambient temperature,eluting with 10 mM Ammonium Bicarbonate in water adjusted to pH 10 withAmmonia solution (Solvent A) and Acetonitrile (Solvent B) using anelution gradient of between 0 and 100% Solvent B over 15 or 25 minutes.

The UV detection was an averaged signal from wavelength of 210 nm to 350nm. The mass spectra were recorded on a Waters ZQ Mass Spectrometerusing Alternate-scan Positive and Negative Electrospray. Ionisation datawas rounded to the nearest integer.

MDAP (Method F). The HPLC analysis was conducted on a Sunfire C18 column(150 mm×30 mm i.d. 5 μm packing diameter) at ambient temperature,eluting with 0.1% v/v solution of Trifluoroacetic Acid in Water (SolventA) and 0.1% v/v solution of Trifluoroacetic Acid in Acetonitrile(Solvent B) using an elution gradient of between 0 and 100% Solvent Bover 15 or 25 minutes.

The UV detection was an averaged signal from wavelength of 210 nm to 350nm. The mass spectra were recorded on a Waters ZQ Mass Spectrometerusing Alternate-scan Positive and Negative Electrospray. Ionisation datawas rounded to the nearest integer.

MDAP (Method G). The HPLC analysis was conducted on a Sunfire C18 column(150 mm×30 mm i.d. 5 μm packing diameter) at ambient temperature,eluting with 0.1% formic acid in water (Solvent A) and 0.1% formic acidin acetonitrile (Solvent B) using an elution gradient of between 0 and100% Solvent B over 15 or 25 minutes.

The UV detection was an averaged signal from wavelength of 210 nm to 350nm. The mass spectra were recorded on a Waters ZQ Mass Spectrometerusing Alternate-scan Positive and Negative Electrospray. Ionisation datawas rounded to the nearest integer.

General Chiral HPLC Methods

Method A: Chiral Analytical Chromatography

Column Chiralpak AD-H, 250 × 4.6 mm Mobile Phase A: n-Hexane B: EthanolGradient Profile 90:10 mobile phase A:B Flow Rate 1 mL/min Column 20° C.Temperature Detection 215 nm or UV DAD (300 nm (bandwidth 180 nm,wavelength reference 550 nm (bandwidth 100 nm))Method B: Chiral Preparative Chromatography

Column Chiralpak AD-H, 250 × 30 mm, 5 μm [ADH10029-01] Mobile Phase A:n-Hexane B: Ethanol Gradient Profile Stepped Isocratic system - 90:10mobile phase A:B Run Time 20 min Flow Rate 45 mL/min Column 20° C.Temperature Detection UV DAD (300 nm (bandwidth 180 nm, reference 550 nm(bandwidth 100 nm))Method C: Chiral Preparative ChromatographyInitial Conditions:

Column Chiralpak AD, 250 × 20 mm, 20 μm [self packed] Mobile Phase A:n-Hexane B: Ethanol Gradient Profile 90:10 mobile phase A:B Flow Rate 75mL/min Column 20° C. Temperature Detection 215 nm wavelength

An initial cut of the leading edge of the peak was taken using theinitial conditions. This gave an enriched cut of the desired firsteluting isomer which was then further purified using the secondaryconditions.

Secondary Conditions:

Column Chiralpak AD-H, 250 × 30 mm, 5 μm [ADH10029-01] Mobile Phase A:n-Hexane B: Ethanol Gradient Profile 90:10 mobile phase A:B Flow Rate 40mL/min Column 20° C. Temperature Detection 215 nm wavelengthMethod D: Chiral Preparative Chromatography

Column Chiralpak AD-H, 25 cm × 30 mm, [ADH10029-01] Mobile Phase A:n-Heptane B: isopropanol Gradient Profile 70:30 mobile phase A:B FlowRate 15 mL/min Column 20° C. Temperature Detection 215 nm wavelength

INTERMEDIATES Intermediate 11-(Phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine

To a solution of 1H-pyrrolo[2,3-b]pyridine (20 g, 169 mmol, (availablefrom, for example Sigma Aldrich) in tetrahydrofuran (THF) (250 mL) wasadded sodium hydride (10.16 g, 254 mmol) portionwise during 5 min undernitrogen at 0° C. The reaction mixture was stirred at 0° C. for 30 min,then benzenesulfonyl chloride was added dropwise under nitrogen at 0° C.then stirred for 2 h at r.t., until the starting material had beencompletely consumed (TLC, EtOAc:PE=1:1). The mixture was poured into H₂O(200 mL) and extracted with EtOAc (3×200 mL). The organic layers werewashed with brine (3×150 mL), dried over Na₂SO₄ and filtered. Thesolvent was evaporated in vacuo to give the crude product, which waspurified by recrystallization with (EtOAc and PE) to give desiredproduct as a white solid (30 g, 69%) LCMS (Method D): Rt=1.76 min,MH⁺=259

Intermediate 2 1-(Phenylsulfonyl)-1H-pyrrolo[2,3-c]pyridine

Prepared similarly to intermediate 1 starting from1H-pyrrolo[2,3-c]pyridine (available from, for example, ApolloScientific Ltd).

1H NMR (DMSO-d6): 9.24 (1H, s, CH), 8.40 (1H, d, CH), 8.11-8.08 (3H, m,CH), 7.82-7.62 (4H, m, CH), 6.95 (1H, d, CH).

Intermediate 3 1-(Phenylsulfonyl)-1H-pyrrolo[3,2-c]pyridine

Prepared similarly to intermediate 1 starting from1H-pyrrolo[3,2-c]pyridine (available from, for example, ApolloScientific Ltd).

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 8.91 (1H, s, CH), 8.46 (1H, d, CH),8.07 (2H, d, CH), 7.96-7.92 (2H, m, CH), 7.74 (1H, t, CH), 7.64 (2H, t,CH), 6.99 (1H, d, CH).

Intermediate 41-(Phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde

To a solution of diisopropylamine (4.13 mL, 0.029 mol) in anhydroustetrahydrofuran (THF) (50 mL) stirred under nitrogen at −78° C. wasadded nBuLi (10.42 mL, 0.026 mol) over 15 min. The reaction mixture wasstirred at −78° C. for 30 min. then warmed to r.t. and stirred for 1 h.To this solution of LDA in anhydrous tetrahydrofuran (THF) (250 mL)stirred under nitrogen at −30° C. was added a solution of1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine (5 g, 19.36 mmol) and TMEDA(4.38 mL, 29.0 mmol) in tetrahydrofuran (THF) (150 mL) dropwise over 15min. The reaction mixture was stirred at −30° C. for 2.5 h, then DMF (3mL, 38.7 mmol) was added dropwise over 1 min. The reaction mixture wasstirred at −30° C. for another 2 h, TLC and LC-MS showed completeconversion. The reaction mixture was quenched with water and partitionedbetween dichloromethane (700 mL) and water (100 mL). The organic phasewas washed with water (3×100 mL), dried over sodium sulfate andevaporated in vacuo to give the crude product as a yellow solid. Thiswas purified by recrystallization (EtOAc and PE) to give the desiredproduct—1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (4.8g, 78%) as a yellow solid.

1H NMR (DMSO-d6): 10.45 (1H, s, CH), 8.58 (1H, dd, CH), 8.24-8.16 (3H,m, CH), 7.74 (1H, t, CH), 7.66-7.58 (3H, m, CH), 7.41 (1H, dd, CH).

Intermediate 51-(Phenylsulfonyl)-1H-pyrrolo[2,3-c]pyridine-2-carbaldehyde

Prepared similarly to intermediate 4 starting from1-(phenylsulfonyl)-1H-pyrrolo[2,3-c]pyridine.

1H NMR (DMSO-d6): 10.43 (1H, s, CH), 8.68 (1H, dd, CH), 8.55 (1H, d,CH), 8.02 (2H, dd, CH), 7.76-7.72 (2H, m, CH), 7.62-7.56 (3H, m, CH).

Intermediate 61-(Phenylsulfonyl)-1H-pyrrolo[3,2-c]pyridine-2-carbaldehyde

Prepared similarly to intermediate 4 starting from1-(phenylsulfonyl)-1H-pyrrolo[3,2-c]pyridine LCMS (Method D): Rt=1.39min, MH⁺=286.9.

Intermediate 7 1 1H-Pyrrolo[2,3-b]pyridine-2-carbaldehyde

To a solution of1-(phenylsulfonyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (2.5 g, 8.73mmol) in methanol (50 mL) stirred under nitrogen at r.t. was added asolution of KOH (1.96 g, 34.9 mmol) in water (5 mL) dropwise during 1min. The reaction mixture was stirred at r.t. for 30 min, TLC showedcomplete conversion. The reaction mixture was diluted with H₂O (150 mL),extracted with dichloromethane (3×150 mL) and the organic phase waswashed with saturated brine (3×50 mL), water 100 mL, dried over sodiumsulfate and evaporated in vacuo to give the title compound as a yellowsolid (1 g, 54.9% yield) which was used in the next reaction withoutfurther purification.

1H NMR (DMSO-d6): 12.51 (1H, br s, NH), 9.90 (1H, s, CH), 8.48 (1H, dd,CH), 8.21 (1H, d, CH), 7.41 (1H, s, CH), 7.20 (1H, dd, CH).

Intermediate 8 1H-Pyrrolo[2,3-c]pyridine-2-carbaldehyde

Prepared similarly to intermediate 7 starting from1-(phenylsulfonyl)-1H-pyrrolo[2,3-c]pyridine-2-carbaldehyde.

1H NMR (DMSO-d6): 12.40 (1H, br s, NH), 9.99 (1H, s, CH), 8.87 (1H, s,CH), 8.19 (1H, d, CH), 7.74 (1H, dd, CH), 7.42 (1H, s, CH).

Intermediate 9 1H-Pyrrolo[3,2-c]pyridine-2-carbaldehyde

Prepared similarly to intermediate 7 starting from1-(phenylsulfonyl)-1H-pyrrolo[3,2-c]pyridine-2-carbaldehyde.

1H NMR (DMSO-d6): 12.34 (1H, brs, NH), 9.94 (1H, s, CH), 9.07 (1H, s,CH), 8.34 (1H, d, CH), 7.57 (1H, s, CH), 7.41 (1H, d, CH).

Intermediate 10 1-Ethyl-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde

To a suspension of 1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (700 mg,4.79 mmol) and Cs₂CO₃ (3121 mg, 9.58 mmol) in N,N-dimethylformamide(DMF) (20 mL) stirred under nitrogen at 20° C., was added iodoethane(0.581 mL, 7.18 mmol) dropwise during 0.5 min. The reaction mixture wasstirred at rt for 1 h, TLC showed complete conversion.

In a separate reaction: To a suspension of1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (600 mg, 4.11 mmol) and Cs₂CO₃(2675 mg, 8.21 mmol) in N,N-Dimethylformamide (DMF) (20 mL) stirredunder nitrogen at 20° C. was added iodoethane (0.498 mL, 6.16 mmol)dropwise during 0.5 min. The reaction mixture was stirred at r.t. for 1h, TLC showed complete conversion.

The combined reaction mixtures were quenched with water, partitionedbetween dichloromethane (100 mL) and water (50 mL). the water phase wasextracted with dichloromethane (3×100 mL). The organic phase was washedwith saturated brine (3×50 mL), dried over sodium sulfate and evaporatedin vacuo to give the crude product as a yellow oil. The crude productwas purified by a silica gel column (Hex/EtOAc, 10/1) to give the titlecompound (511 mg, 32%).

LCMS (Method E): Rt=1.45 min, MH⁺=175.1.

Intermediate 11 1-Ethyl-H-pyrrolo[2,3-c]pyridine-2-carbaldehyde

Prepared similarly to intermediate 10 starting from1H-pyrrolo[2,3-c]pyridine-2-carbaldehyde GCMS: Rt=14.32 min, M⁺=174.

Intermediate 12 1-Ethyl-1H-pyrrolo[3,2-c]pyridine-2-carbaldehyde

Prepared similarly to intermediate 10 starting from1H-pyrrolo[3,2-c]pyridine-2-carbaldehyde.

LCMS (Method E): Rt=0.61 min, MH⁺=175.1.

Intermediate 131-(Cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde

A solution of sodium hydride (60.2 mg, 1.51 mmol) inN,N-dimethylformamide (20 mL) was stirred at 0° C. for 10 min.1H-Pyrrolo[2,3-b]pyridine-2-carbaldehyde (200 mg, 1.37 mmol) was addedand the mixture was stirred at 0° C. for 30 min and at rt for 30 min.(Bromomethyl)cyclopropane (0.16 mL, 1.64 mmol available, for example,from Alfa Aesar) was added and the resulting mixture was stirred at 0°C. for 30 min and at rt for 21 h. Reaction mixture was quenched by theaddition of water (50 mL). After addition of Et₂O (50 mL), the layerswere separated. The aqueous layer was further extracted with Et₂O (2×50mL) and the combined organic layers were washed with H₂O (2×35 mL). Theorganic phase was dried through a hydrophobic frit and concentratedunder reduced pressure to give a brown oil which was loaded in DCM on a50 g SNAP silica cartridge and purified by SP4, eluting with a gradientof 0-20% ethyl acetate/cyclohexane (15 CV). The appropriate fractionswere combined and evaporated under reduced pressure to give the requiredproduct 1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde(201 mg, 73.4%) as a colourless oil.

LCMS (Method B): Rt=0.99 min, MH⁺=201.0

Intermediate 141-(2,2,2-Trifluoroethyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde

1H-Pyrrolo[2,3-b]pyridine-2-carbaldehyde (1.8 g, 12.32 mmol) was addedto a solution of sodium hydride (0.54 g, 13.55 mmol) inN,N-dimethylformamide (50 mL) at rt under nitrogen. The reaction mixturewas allowed to stir for 1 h before the reaction mixture was cooled to 0°C. and 2,2,2-trifluoroethyl trifluoromethanesulfonate (2.04 ml, 14.78mmol available, for example, from Sigma Aldrich) was added dropwise. Thereaction mixture was stirred for 1 h at 0° C. and for 14 h at rt undernitrogen. The reaction was quenched by addition of water (150 mL). Afteraddition of Et₂O (150 mL), the layers were separated. The aqueous layerwas further extracted with Et₂O (3×150 mL) and the combined organiclayers were washed with H₂O. The combined water layers were extractedwith Et₂O (150 mL). The organic layers collected were combined, driedover Na₂SO₄, filtered and concentrated under reduced pressure. Theresidue was loaded in dichloromethane on two 100 g SNAP silica cartridgeand purified by SP4, eluting with 0-30% ethyl acetate/cyclohexanegradient. The appropriate fractions were combined and evaporated underreduced pressure to give the required product1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (2.6g, 11.39 mmol, 93% yield) as a white solid.

LCMS (Method B): Rt=0.92 min, MH⁺=229.14.

Intermediate 15 Ethyl1-ethyl-5-methoxy-1H-pyrrolo[2,3-b]pyridine-2-carboxylate

To a solution of ethyl 5-methoxy-1H-pyrrolo[2,3-b]pyridine-2-carboxylate(150 mg, 0.68 mmol available, for example, from Shanghai HaoyuanChemexpress Co., Ltd) in DMSO (4 ml) was added powdered potassiumhydroxide (115 mg, 2.04 mmol) followed by bromoethane (0.071 ml, 0.95mmol). The mixture was stirred at rt for 2 hr then further bromoethane(0.020 ml) was added and the mixture stirred for a further 18 hr. Thereaction was quenched by the addition of water then partitioned betweenwater and diethyl ether. The organic phase was washed with water thenpassed through a hydrophobic frit and finally concentrated under reducedpressure to give the product as an orange/brown oil. The crude materialwas purified with column chromatography (eluted with DCM and ethylacetate from 0 to 10%) to give the title compound as clear oil (60 mg,36%).

LCMS (Method C): MH⁺=249.1, Rt=1.15 min

Intermediate 16 1-Ethyl-5-methoxy-1H-pyrrolo[2,3-b]pyridine-2-carboxylicacid

To a solution of ethyl1-ethyl-5-methoxy-1H-pyrrolo[2,3-b]pyridine-2-carboxylate (60 mg, 0.24mmol) in THF (2 ml) and MeOH (0.5 ml) was added lithium hydroxide (35mg, 1.45 mmol) in water (2 mL). A cloudy solution formed which becameclear after 30 s. The reaction mixture was allowed to stand at roomtemperature for 18 hr then concentrated under a stream of nitrogen. 2MHCl (2 ml, aqueous) was added to the crude product and the resultingsolid filtered then dried under reduced pressure to give the titlecompound as an off-white solid (32 mg, 60%).

LCMS (Method C): Rt=0.80 min, MH⁺=221.1

Intermediate 17 1-Ethyl-5-methoxy-1H-pyrrolo[2,3-c]pyridine-2-carboxylicacid

To a solution of 5-methoxy-1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid(500 mg, 2.60 mmol) (available from, for example, Activate ScientificGmbH) in dimethyl sulfoxide (DMSO) (5 mL) was added potassium hydroxide(438 mg, 7.81 mmol) and bromoethane (0.427 mL, 5.72 mmol). The reactionwas stirred at room temperature for 18 h before adding further potassiumhydroxide (120 mg) and bromoethane (0.13 ml). After stirring for afurther 4 h, the reaction mixture was stood under nitrogen for ca. 66 h.It was then partitioned between water and diethyl ether. The layers wereseparated and the aqueous was washed with diethyl ether. The aqueous wasacidified to pH=3, and was extracted with ethyl acetate (twice). Thecombined ethyl acetate extracts were washed with water and concentratedin vacuo to yield the title compound as a pale beige solid (148 mg).

LCMS (Method C): Rt=0.46 min, MH⁺=221

Intermediate 18 Methyl 4-(methylamino)-3-nitrobenzoate

Methylamine (2M in THF) (23.19 mL, 46.4 mmol) was added to a solution ofmethyl 4-chloro-3-nitrobenzoate (5 g, 23.19 mmol) (available, forexample, from Lancaster Synthesis Ltd.) in N,N-dimethylformamide (DMF)(8 mL) at rt under nitrogen. The reaction mixture was heated to 80° C.and stirred overnight. LCMS showed major peak product, but reaction hadnot gone to completion. Further methylamine (2M in THF, 10 ml) was addedand the reaction heated to 90° C. for 6 h. Further methylamine (2M inTHF, 6 ml) was added and the reaction stirred for 1 h at rt and 72 h at70° C. Further methylamine (2M in THF, 10 ml) was added and the reactionheated to 80° C. for 3 h. The reaction was allowed to cool to rt andthen the product was precipitated by the addition of water (50 mL). Theresultant suspension was cooled to 0° C. and then filtered. The residuewas washed with further water (3×25 mL) and allowed to dry on the filterpad for −15 mins. The solid was collected and dried in vacuo to affordthe title compound as a yellow solid (4.54 g, 21.60 mmol, 93% yield).

LCMS (Method B): Rt=0.69 min, MH⁺=197.2

Intermediate 19 4-(Methylamino)-3-nitrobenzoic acid

Methyl 4-(methylamino)-3-nitrobenzoate (1.82 g, 8.66 mmol) was dissolvedin a 1:1 ratio of tetrahydrofuran (THF) (41.4 mL) and water (41.4 mL).To this was added lithium hydroxide (1.817 g, 43.3 mmol) and thereaction stirred at r.t. for 16 h. The reaction mixture was cooled to 0°C. and acidified by the addition of 5M HCl (˜20 mL, until the pH reached˜5)—a bright yellow precipitate formed, the slurry was filtered and theresidue washed with distilled H₂O (2×30 mL). The residue was collectedand dried in vacuo at 50° C. to afford the product as a yellow solid(1.43 g, 7.29 mmol, 84% yield). This was used without furtherpurification in the subsequent reactions.

LCMS (Method B): Rt=0.92 min, MH⁺=211

Intermediate 20(R)-tert-Butyl(1-(4-(methylamino)-3-nitrobenzol)piperidin-3-yl)carbamate

To a solution of (R)-tert-butyl piperidin-3-ylcarbamate (1.460 g, 7.29mmol) (available from, for example, Apollo Scientific Ltd),4-(methylamino)-3-nitrobenzoic acid (1.43 g, 7.29 mmol) and HATU (2.77g, 7.29 mmol) in N,N-dimethylformamide (DMF) (50 mL) was added DIPEA(2.55 mL, 14.58 mmol) and the reaction stirred at r.t. for 16 h. Water(200 mL) and Et₂O (200 mL) were added and the layers separated. Theaqueous layer was extracted with further Et₂O (2×200 mL) and thecombined organics washed with water (2×50 mL), dried (Na₂SO₄) andconcentrated in vacuo to afford a bright yellow oil. The crude productwas purified on silica (100 g) using a gradient of 40%EtOAc/cyclohexane→100% ethyl acetate/cyclohexane. The appropriatefractions were combined and evaporated under vacuum to give the titleproduct as an orange-gold solid (2.76 g, 7.29 mmol, 100% yield).

LCMS (Method B): Rt=0.96 min, MH⁺=379.3

Intermediate 21tert-Butyl(1-(4-(methylamino)-3-nitrobenzoyl)piperidin-3-yl)carbamate

Prepared similarly to intermediate 20 from tert-butylpiperidin-3-ylcarbamate (available from, for example, Apollo ScientificLtd) and 4-(methylamino)-3-nitrobenzoic acid.

LCMS (Method B): Rt=0.96 min, MH⁺=379.2

Intermediate 22(R)-tert-Butyl(1-(3-amino-4-(methylamino)benzoyl)piperidin-3-yl)carbamate

(R)-tert-Butyl(1-(4-(methylamino)-3-nitrobenzoyl)piperidin-3-yl)carbamate(3.79 g, 10.02 mmol) was dissolved in ethanol (75 mL) and added to aflushed hydrogenation flask containing Pd/C (380 mg, 0.411 mmol). Theresultant mixture was flushed with nitrogen/vacuum 3 times, then stirredunder an atmosphere of hydrogen at room temperature for 24 hours. Thereaction mixture was flushed from hydrogen atmosphere withnitrogen/vacuum three times. To this solution Celite (33 g) was addedand stirred for 2 min. then filtered under vacuum. The solution wasconcentrated under vacuum to give a crude product that was purified on a100 g SNAP cartridge using SP4 column chromotography. The column waseluted with 0-6% 2M NH₃ in MeOH in DCM over 25 CV. The appropriatefractions were combined and concentrated in vacuo to give a product thatwas further purified using SP4 column chromotography on a 100 g SNAPcartridge. The column was eluted with 0-100% EtOAc in cyclohexane over15 CV followed by 100% EtOAc 5 CV followed by 0-6% 2M NH3 in MeOH in DCMover 15 CV. The appropriate fractions were combined and concentrated invacuo to give the title compound as a pink solid (1.26 g).

LCMS (Method B): Rt=0.70 min, MH⁺=349.1

Intermediate 23 Methyl 3-methoxy-4-(methylamino)-5-nitrobenzoate

Methyl 4-chloro-3-methoxy-5-nitrobenzoate (available from, for example,Apollo Scientific Ltd) (14 g, 57.0 mmol) was dissolved inN,N-dimethylformamide (DMF) (140 mL) and cooled to −0° C. in anice/water bath. Methanamine (2M in THF) (114 mL, 228 mmol) was addeddropwise with vigorous stirring using a dropping funnel and the mixturewas flushed with nitrogen and heated at 80° C. for 3 hr. The mixture wasallowed to cool to room temperature over the weekend. The reactionmixture was diluted with water (500 mL), and filtered under vacuum togive the title compound as an orange solid (13.69 g).

LCMS (Method A): Rt=1.04 min, MH⁺=241.05

Intermediate 24 3-Methoxy-4-(methylamino)-5-nitrobenzoic acid

To a solution of methyl 3-methoxy-4-(methylamino)-5-nitrobenzoate (13.69g, 57.0 mmol) in tetrahydrofuran (THF) (100 mL) and water (50.0 mL) wasadded a single portion of lithium hydroxide (4.09 g, 171 mmol). Theresulting suspension was stirred for 19 hr at room temperature. Thereaction was acidified with aq. 2N HCl (˜50 mL), until pH reached ˜4.The resultant suspension was filtered and the orange solid dried on thehigh vacuum line overnight to give the title compound as an orange solid(11.09 g).

LCMS (Method A): Rt=0.51 min, MH⁺=227.0

Intermediate 25(R)-tert-Butyl(1-(3-methoxy-4-(methylamino)-5-nitrobenzoyl)piperidin-3-yl)carbamate

To a solution of 3-methoxy-4-(methylamino)-5-nitrobenzoic acid (11.09 g,49.0 mmol) and HATU (18.64 g, 49.0 mmol) in N,N-dimethylformamide (DMF)(300 mL) was added DIPEA (17.13 mL, 98 mmol) and the mixture stirred for30 mins. Upon addition of the DIPEA the mixture went cloudy after ˜1 minwith stirring. (R)-tert-Butyl piperidin-3-ylcarbamate (9.82 g, 49.0mmol) was then added and stirred for 1.5 hr, after which time LCMSshowed the reaction was complete. To 5 mL of the reaction mixture wasadded sat.aq. LiCl solution (5 mL) and Et₂O (10 mL) and the layersseparated. The aqueous layer was re-extracted with Et₂O (2×10 mL), thecombined organics were backwashed with water (10 mL), dried with Na₂SO₄,filtered and concentrated in vacuo to give the crude product as anorange gum. The gum was dissolved in the minimum amount of DCM andpurified by Si SNAP 25 g column using a 50-100% ethylacetate/cyclohexane. The appropriate fractions were combined andevaporated in vacuo before being azeotroped with cyclohexane and driedunder vacuum to give the required product, 281 mg as an orange solid.The remaining reaction mixture was concentrated in vacuo to remove someof the DMF. Saturated aq.LiCl solution (300 mL) and Et₂O (700 mL) wereadded and the mixture separated. The aqueous layer was re-extracted withEt₂O (2×700 mL), the combined organic layers were backwashed with water(1 L), dried with Na₂SO₄, filtered and concentrated in vacuo to give thecrude product as an orange gum. This was purified on a 340 g SNAP silicacartridge eluting with 30%-60% ethyl acetate in cyclohexane. Appropriatefractions were combined and concentrated in vacuo to yield the titlecompound as an orange solid (19.4 g).

LCMS: (Method B): Rt=1.02 min, MH⁺=409.1

Intermediate 26(R)-tert-Butyl(1-(2-(1-ethyl-1H-pyrrolo[2,3-c]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate

Sodium hydrosulfite (162 mg, 0.793 mmol) dissolved in water (1 mL) wasadded to a solution of(R)-tert-butyl(1-(4-(methylamino)-3-nitrobenzoyl)piperidin-3-yl)carbamate(100 mg, 0.264 mmol) and1-ethyl-1H-pyrrolo[2,3-c]pyridine-2-carbaldehyde (46.0 mg, 0.264 mmol)in ethanol (2 mL) at RT under nitrogen. The reaction mixture was heatedto 100° C. in a microwave for 5 h. The reaction mixture was diluted withDCM (20 mL), sodium sulfate was added and the resultant suspensionfiltered and concentrated in vacuo to yield the crude product. This waspurified by Biotage SP4 on a SNAP 10 g silica cartridge using a gradientof 0% (20% MeOH/DCM)/DCM→50% (20% MeOH/DCM)/DCM. Appropriate fractionswere combined and evaporated under vacuum to give the title compound (42mg)

LCMS (Method A): Rt=0.71 min, MH⁺=503.3

Intermediate 27tert-Butyl(1-(2-(1-ethyl-1H-pyrrolo[3,2-c]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate

Sodium hydrosulfite (277.4 mg, 1.275 mmol) dissolved in water (1.5 mL)was added to a stirred solution oftert-butyl(1-(4-(methylamino)-3-nitrobenzoyl)piperidin-3-yl)carbamate(143.2 mg, 0.378 mmol) and1-ethyl-1H-pyrrolo[3,2-c]pyridine-2-carbaldehyde (67.6 mg, 0.388 mmol)in ethanol (3.5 mL) at room temperature in a 5 ml microwave vial. Thereaction mixture was then heated in a microwave for 5 hr at 100° C.Methanol was added to the reaction mixture and dried using Na₂SO₄. Thereaction mixture was then filtered under gravity through a hydrophobicfrit and the eluent collected and concentrated under vacuum. The crudeproduct was dissolved in a minimum volume of DCM and purified using SP4on a 25 g SNAP silica cartridge. The cartridge was eluted using agradient of 0-100% 20% methanol in DCM/DCM. Appropriate fractions werecollected and concentrated under vacuum to give the title compound as ayellow oil (76 mg).

LCMS (Method B): Rt=0.71 min, MH⁺=503.2

Intermediate 28(R)-tert-Butyl(1-(1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate

To a mixture of(R)-tert-butyl(1-(4-(methylamino)-3-nitrobenzoyl)piperidin-3-yl)carbamate(2.1 g, 5.55 mmol) and1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (1.3g, 5.70 mmol) in ethanol (55 ml) was added portionwise a solution ofsodium hydrosulfite (3.41 g, 16.65 mmol) in water (25 ml). The mixturewas flushed with nitrogen and heated at 90° C. overnight for 17 hours.The reaction mixture was allowed to cool to room temperature and thesolvent was evaporated in vacuo. DCM was added to the residue and theheterogeneous solution was dried over sodium sulfate. The solid wasfiltered off and the filtrate was concentrated under vacuo. The residuewas loaded in dichloromethane and purified by SP4 SNAP on 2 silica (Si)100 g columns using an initial gradient of 25-80% (15 CVs), followed by80-100% (10 CVs) ethyl acetate/cyclohexane. The appropriate fractionswere combined and evaporated in vacuo to give the title compound as apale yellow solid (1.934 g).

LCMS (Method B): Rt=1.09 min, MH⁺=557.5

Intermediate 29(R)-tert-Butyl(1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate

A solution of1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (2 g,8.99 mmol) in ethanol (90 mL) was added to a round bottom flaskcontaining(R)-tert-butyl(1-(4-(methylamino)-3-nitrobenzoyl)piperidin-3-yl)carbamate(3.4 g, 8.98 mmol) and the resulting solution stirred at roomtemperature. A solution of sodium dithionite (3.14 g, 15.33 mmol) inwater (45 mL) was added portionwise to the reaction mixture. Thereaction mixture was heated to 100° C. and stirred under nitrogen for 4hours. The reaction mixture was concentrated under vacuum then dilutedwith DCM (150 ml) and water (150 ml). The organic layer was collectedand the aqueous layer washed with DCM (3×100 ml). Organic layers werecollected and backwashed with water (2×100 ml). The organic layer wascollected, dried with Na₂SO₄, filtered through a hydrophobic frit andconcentrated under vacuum to yield a crude product. This was dissolvedin a minimum volume of DCM and purified using Biotage SP4 on a 100 gSNAP silica cartridge. The column was eluted with a gradient of 70-100%ethyl acetate in DCM for 10 CV. Appropriate fractions were collected andconcentrated under vacuum. This was dried under vacuum to yield thetitle compound as a yellow solid (1.867 g).

LCMS (Method B): Rt=1.08 min, MH⁺=529.4

Intermediate 30(R)-tert-Butyl(1-(2-(1-ethyl-5-methoxy-1H-pyrrolo[2,3-c]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate

1-Ethyl-5-methoxy-1H-pyrrolo[2,3-c]pyridine-2-carboxylic acid (200 mg,0.908 mmol, reported in WO 2010/118208) and HATU (380 mg, 0.999 mmol)were dissolved in DMF (2 mL) and stirred at rt for 5 min. To this wasadded a solution of(R)-tert-butyl(1-(4-(methylamino)-3-nitrobenzoyl)piperidin-3-yl)carbamate(316 mg, 0.908 mmol) and DIPEA (0.476 ml, 2.72 mmol) in DMF (2 ml), andthe resulting mixture stirred under nitrogen at rt for 3.5 hr. Thereaction mixture was diluted with water (40 ml) and partitioned withether (50 ml). The organic layer was isolated then the aqueous layerre-extracted with ether (2×50 ml). Combined organic layers were washedwith water (2×30 ml) then dried over sodium sulfate then passed througha hydrophobic frit and concentrated under reduced pressure to give thecrude amide intermediate as a blue solid. The solid was dried underreduced pressure overnight then dissolved in toluene (12.5 ml). Aceticacid (0.052 ml, 0.908 mmol) was added to the reaction mixture which wasrefluxed for 5 hr. Sodium bicarbonate (40 ml) was added to the reactionmixture and the organic layer isolated. The aqueous layer wasreextracted with toluene (2×40 ml) and combined organic layers wereconcentrated under reduced pressure to give 238 mg of the crude productas a red-brown gum. The crude material was purified with columnchromatography (eluted with 100% EtOAc) then further purified by high pHMDAP (Method E) to give the title compound as an off-white solid (106mg, 22%).

LCMS (Method A): MH⁺=533.4, Rt=1.06 min

Intermediate 31(R)-tert-Butyl(1-(2-(1-ethyl-5-methoxy-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate

Prepared similarly to intermediate 30 starting from1-ethyl-5-methoxy-1H-pyrrolo[2,3-b]pyridine-2-carboxylic acid.

LCMS (Method A): Rt=1.10 min, MH⁺=533.3

Intermediate 32(R)-tert-Butyl(1-(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate

To a solution of(R)-tert-butyl(1-(3-methoxy-4-(methylamino)-5-nitrobenzoyl)piperidin-3-yl)carbamate(4.5 g, 11.02 mmol) and 1-ethyl-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde(2.015 g, 11.57 mmol) in ethanol (100 mL) was added portionwise asolution of sodium dithionite (4.25 g, 20.75 mmol) in water (50 mL). Themixture was flushed with nitrogen then heated at 100° C. overnight (16hours). The reaction mixture was concentrated under vacuum then dilutedwith DCM (150 ml) and water (150 ml). The organic layer was collectedand the aqueous layer washed with DCM (3×100 ml). Organic layers werecombined and back washed with water (3×150 ml), collected, dried withNa₂SO₄, filtered through a hydrophobic frit and concentrated undervacuum to yield 5.5 g of crude product as white solid. The crude productwas dissolved in a minimum volume of DCM and purified using Biotage SP4on a SNAP 100 g silica cartridge. The column was eluted with a gradientof 70-100% ethyl acetate in DCM for 10 CV. Appropriate fractions werecollected and concentrated in vacuo to afford the title compound as awhite solid (4.40 g).

LCMS (Method B): Rt=1.05 min, MH⁺=533.4

Intermediate 33(R)-tert-Butyl(1-(7-methoxy-1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridine-2-yl)-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate

To a solution of(R)-tert-butyl(1-(3-methoxy-4-(methylamino)-5-nitrobenzoyl)piperidin-3-yl)carbamate(3.58 g, 8.76 mmol) and1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (2.61g, 11.44 mmol) in ethanol (140 ml) was added portionwise a solution ofsodium hydrosulfite (3.18 g, 15.53 mmol) in water (70 ml). The mixturewas flushed with nitrogen then heated at 100° C. overnight (16 hours).The reaction mixture was concentrated under vacuum then diluted with DCM(150 ml) and water (150 ml). The organic layer was collected and theaqueous layer washed with DCM (3×100 ml). Organic layers were collected,dried with Na₂SO₄, filtered through a hydrophobic frit and concentratedunder vacuum to yield 5.5 g of crude product as white solid. This wasdissolved in a minimum volume of DCM and purified using Biotage SP4 on a100 g SNAP silica cartridge. The column was eluted with a gradient of70-100% ethyl acetate in DCM for 10 CV. Appropriate fractions werecollected and concentrated under vacuum to yield the title compound as awhite solid (4.31 g).

LCMS (Method B): Rt=1.17 min, MH⁺=587.4

Intermediate 34(R)-tert-Butyl(1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate

A solution of1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (0.743 g,3.71 mmol) in ethanol (60 mL) was added to a round bottom flaskcontaining(R)-tert-butyl(1-(3-methoxy-4-(methylamino)-5-nitrobenzoyl)piperidin-3-yl)carbamate(1.529 g, 3.74 mmol) and the resulting solution stirred at roomtemperature. A solution of sodium dithionite (1.375 g, 6.71 mmol) inwater (30 mL) was added portionwise to the reaction mixture. Thereaction mixture was heated to 100° C. and stirred under nitrogen for 4hours. The reaction mixture was concentrated under vacuum then dilutedwith DCM (150 ml) and water (150 ml). The organic layer was collectedand the aqueous layer washed with DCM (3×100 ml). Organic layers werecollected, dried with Na₂SO₄, filtered through a hydrophobic frit andconcentrated under vacuum to yield ˜2 g of crude product as yellowsolid. This was dissolved in a minimum volume of DCM and purified usingBiotage SP4 on a 50 g SNAP silica cartridge. The column was eluted witha gradient of 70-100% ethyl acetate in DCM for 10 CV. Appropriatefractions were collected and concentrated under vacuum to yield thetitle compound as a yellow solid (1.55 g).

LCMS (Method B): Rt=1.15 min, MH⁺=559.4

Intermediate 35tert-Butyl(1-(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate

Sodium hydrosulfite (235 mg, 1.150 mmol) dissolved in water (1.500 mL)was added to a solution oftert-butyl(1-(4-(methylamino)-3-nitrobenzoyl)piperidin-3-yl)carbamate(145 mg, 0.383 mmol) and1-ethyl-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (66.7 mg, 0.383 mmol)in ethanol (3 mL) at RT under nitrogen. The reaction mixture was heatedto 100° C. in a microwave for 5 h. The reaction mixture was diluted withDCM (20 mL), Na₂SO₄ was added and the resultant suspension filtered andconcentrated in vacuo to yield the crude product as a yellow oil. Thecrude product was purified by Biotage SP4 on a SNAP 25 g silicacartridge using a gradient of 0% (20% MeOH/DCM)/DCM→100% (20%MeOH/DCM)/DCM. Appropriate fractions were combined and evaporated undervacuum to give the title compound as a yellow solid (104 mg).

LCMS (Method B): Rt=1.01 min, MH⁺=503.2

Intermediate 36 Benzyl 7-oxa-3-azabicyclo[4.1.0]heptane-3-carboxylate

3-Chlorobenzoperoxoic acid (16.79 g, 97 mmol) was added portionwiseunder an atmosphere of nitrogen to a stirred solution of benzyl5,6-dihydropyridine-1(2H)-carboxylate (15.1 g, 69.5 mmol) (available,for example, from Fluorochem) in anhydrous dichloromethane (DCM) (100mL) cooled using an ice bath. The resulting mixture was allowed to reachrt and stirred for 18 h. Water (100 mL) was added to the reactionmixture and the layers were partitioned. The organic layer was addeddropwise to a stirred 5% aqueous solution of NaS₂O₅ (200 mL). At the endof the addition, the mixture was stirred for a further 1 h, then thelayers were separated and the aqueous layer was back extracted with DCM(50 mL×2). The organics were combined and washed with 5% aqueous K₂CO₃solution (100 mL×3), followed by brine (100 mL). At this stage peroxidetest showed there was still 25 mg/mL peroxide in the organic layer. Theorganics were therefore added to a stirred solution of 5% NaS₂O₅ (aq)(200 mL) and the resultant biphasic mixture stirred for 1 h. Peroxidetest now showed <0.5 mg/mL peroxide. The layers were separated andaqueous layer washed with further DCM (2×50 mL). The combined organicswere then dried (Na₂SO₄). and concentrated in vacuo to afford the crudeproduct as a pale-gold oil. The crude product was purified by silica gelchromatography, (340 g Si), eluting with 30→80% EtOAc/cyclohexane. Theappropriate fractions were combined and concentrated in vacuo to affordthe title compound as a colourless oil—benzyl7-oxa-3-azabicyclo[4.1.0]heptane-3-carboxylate (12.75 g, 54.7 mmol, 79%yield).

LCMS (Method B): Rt=0.88 min, MH⁺=234.2

Intermediate 37 trans-Benzyl3-((tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate

Three separate reactions were performed under the same reactionconditions outlined below. Where reagent/solvent quantities vary, thespecific quantities used are outlined in the table. The crude materialfrom the three reactions was combined for purification as indicated:

Reagent/Solvent: Reaction 1 Reaction 2 Reaction 3 Benzyl 7-oxa-3- 4.37g, 4.45 g, 3.94 g, azabicyclo[4.1.0]heptane- 18.73 mmol 19.08 mmol 16.89mmol 3-carboxylate (A) DCM (B) 120 mL 100 ml 100 ml Triethylamine (C)2.87 ml, 2.92 ml, 2.59 mL, 20.61 mmol 20.98 mmol 18.58 mmol Boc₂O (D)4.35 ml, 4.43 ml, 3.92 mL, 18.73 mmol 19.08 mmol 16.89 mmol

A solution of benzyl 7-oxa-3-azabicyclo[4.1.0]heptane-3-carboxylate (A)in 25-30% ammonium hydroxide aqueous solution (150 ml, 3766 mmol) andethanol (100 mL) was stirred in a HASTC alloy bomb at 70° C. for 5 h.The reaction mixture was transferred to a rb flask and concentrated invacuo by half (caution large amount of NH₃ given off). The resultantsolution was diluted with brine (50 mL) and the organics extracted intoDCM (100 mL). Subsequently the aqueous layer was further extracted with10% MeOH/DCM (3×50 mL). The combined organic layers were dried (Na₂SO₄)and concentrated in vacuo to give the intermediate primary amine as ayellow oil. The oily residue was diluted with dichloromethane (DCM) (B)and triethylamine (C) and Boc₂O (D) added dropwise. The reaction wasallowed to stir for 2 h. LCMS showed complete reaction to two regiomericproducts with similar Rt. The reaction mixture was quenched with sat.NH₄Cl (aq) (100 mL) and the layers separated. The aqueous was furtherextracted with DCM (2×75 mL). The combined organics were dried through ahydrophobic frit and the solvent was removed under vacuum to give awhite gum.

The crude material from the three reactions was combined forpurification: The combined residue was dissolved in DCM and split in twoand purified by column chromatography on two 340 g silica cartridges,using a gradient of 0-100% ethyl acetate/cyclohexane. The appropriatefractions were combined and evaporated in vacuo to give two mainproducts:

First eluting peak from column: trans-benzyl4-((tert-butoxycarbonyl)amino)-3-hydroxypiperidine-1-carboxylate (10.492g, 29.9 mmol, 59% yield) as a white solid (undesired regioisomer).

Second eluting peak from column: trans-benzyl3-((tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate (6.485g, 18.51 mmol, 37% yield) as a white solid (desired regioisomerindicated above.)

LCMS (Method B): Rt=0.96 min, MH⁺=351.2

Intermediate 38 cis-Benzyl4-(benzoyloxy)-3-((tert-butoxycarbonyl)amino)piperidine-1-carboxylate

To a solution of triphenylphosphine (5.83 g, 22.24 mmol) intetrahydrofuran (THF) (60 mL) was added DIAD (4.38 mL, 22.24 mmol) andthe mixture was stirred in an ice-water bath for 15 min and then allowedto warm to rt. To the suspension was added a suspension of trans-benzyl3-((tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate (6.495g, 18.54 mmol) in tetrahydrofuran (THF) (75 mL) followed by benzoic acid(2.72 g, 22.24 mmol). The reaction mixture cleared to a yellow solutionand was stirred for 2 h. LCMS analysis showed product formation, howeverthe SM peak was obscured by by-product so it was difficult to confirmreaction had gone to completion. The reaction was left to stir overnight(20 h). The reaction mixture was concentrated under vacuum. The residuewas purified by silica chromatography. The residue was loaded in DCM ona 340 g silica cartridge and purified using a 0-40% EtOAc/cyclohexanegradient. The appropriate fractions were combined and the evaporated invacuo to give the crude product cis-benzyl4-(benzoyloxy)-3-((tert-butoxycarbonyl)amino)piperidine-1-carboxylate(8.11 g, 17.84 mmol, 96% yield) as a pale yellow oil.

LCMS (Method B): Rt=1.27 min, MH⁺=455.3.

Intermediate 39 cis-Benzyl3-((tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate

Intermediate 40 (3S,4R)-Benzyl3-((tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate

Intermediate 41 (3R,4S)-Benzyl3-((tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate

A solution of potassium carbonate (3.70 g, 26.8 mmol) in water (80 mL)was added to a solution of cis-benzyl4-(benzoyloxy)-3-((tert-butoxycarbonyl)amino)piperidine-1-carboxylate(8.11 g, 17.84 mmol) in ethanol (160 mL) and the mixture was stirred at70° C. for 20 h. The reaction mixture was concentrated in vacuo to ⅓rdvolume and the resultant suspension was diluted with water (50 mL) andextracted using DCM (3×70 mL). The collected organics were combined anddried (Na₂SO₄) and concentrated in vacuo to afford the crude product asa colourless oil. The crude product was then purified by columnchromatography on a silica cartridge (340 g) using a 0-100% ethylacetate/cyclohexane gradient. The appropriate fractions were combinedand evaporated in vacuo to give the required product cis-benzyl3-((tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate (5.54g, 15.81 mmol, 89% yield) as a white foam.

LCMS (Method B): Rt=0.98 min, MH⁺=351.2

1 g of the racemic product was submitted for chiral purificationchromatography using Chiral HPLC Method B. The isomers were successfullyresolved:

Isomer 1, was obtained as a colourless oil—(3S,4R)-benzyl3-((tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate (405mg, 1.156 mmol, 6.48% yield).

LCMS (Method B): Rt=0.97 min, MH⁺=351.2 Chiral HPLC (Method A): 100% ee.

Isomer 2, was obtained as a colourless oil—(3R,4S)-benzyl3-((tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate (411mg, 1.173 mmol, 6.57% yield).

LCMS (Method B): Rt=0.99 min, MH⁺=351.2 Chiral HPLC (Method A): 95% ee.

The remaining 4.5 g of racemate was also submitted for chiralpurification using Chiral HPLC Method C. The isomers were successfullyresolved:

Isomer 1, was obtained as a colourless oil—(3S,4R)-benzyl3-((tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate (1.94g, 5.54 mmol, 31.0% yield).

LCMS (Method B): Rt=0.98 min, MH⁺=351.2 Chiral HPLC (Method A): 98.7%ee.

Isomer 2, obtained as a colourless oil—(3R,4S)-benzyl3-((tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate (1.92g, 5.48 mmol, 30.7% yield).

LCMS (Method B): Rt=0.97 min, MH⁺=351.1 Chiral HPLC (Method A): 96.3%ee.

Intermediate 42 tert-Butyl((3S,4R)-4-hydroxypiperidin-3-yl)carbamate

A solution of (3S,4R)-benzyl3-((tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate (1.94g, 5.54 mmol) in ethanol (48 mL) was added to a hydrogenation flaskcontaining 10% Pd/C (0.059 g, 0.554 mmol) that had been evacuated andback-filled with N₂ (×3). The flask was again evacuated and thenback-filled with H₂ (×3). Enough H₂ to allow complete reaction was thenintroduced to a burette and the system closed and the flask allowed tostir under a H₂ atmosphere overnight. The reaction mixture was filteredthrough Celite and washed with EtOH (2×20 mL) and ethyl acetate (2×20mL). The combined filtrate was concentrated in vacuo to afford theproduct as a cream oilysolid—tert-butyl((3S,4R)-4-hydroxypiperidin-3-yl)carbamate (1.13 g, 5.22mmol, 94% yield).

LCMS (Method B): Rt=0.40 min, MH⁺=217.1

Intermediate 43 tert-Butyl((3R,4S)-4-hydroxypiperidin-3-yl)carbamate

A solution of benzyl3-((tert-butoxycarbonyl)amino)-4-hydroxypiperidine-1-carboxylate (141mg, 0.402 mmol) in methanol (8.05 mL) was hydrogenated using the H-cube(settings: 25° C., full H₂ mode, 1 mL/min flow rate) and 10% Pd/CCatCart 30 as the catalyst. The eluent was evaporated in vacuo to givethe required tert-butyl(4-hydroxypiperidin-3-yl)carbamate (85.1 mg,0.393 mmol, 98% yield) as a clear oil.

¹H NMR (DMSO-d6, 393K): 5.60 (1H, br s, NH), 3.77 (1H, dt, CH), 3.45(1H, ddd, CH), 2.80 (1H, ddd, CH_(A)H_(B)), 2.72 (1H, dd, CH_(A)H_(B)),2.63 (1H, dd, CH_(A)H_(B)), 2.55-2.48 (1H, obs, CH_(A)H_(B)), 1.59-1.53(2H, m, CH₂), 1.42 (9H, s, 3×CH₃).

Proof of Absolute Stereochemistry for Intermediates 42 and 43

The absolute configuration of intermediates 42 and 43 was assigned usingab initio VCD analysis. The confidence level for this assignment wasestimated to be >99%.

Theoretical Analysis:

-   -   Conformational Search: MOE stochastic csearch using MMFF94×        force field    -   Model Chemistry: #opt freq=(noraman, vcd) b3lyp/dgdzvp    -   Conformational Analysis: Fractional populations estimated using        Boltzmann statistics    -   Lorentzian band width: 6 cm⁻¹    -   Frequency scale factor: 0.975    -   Estimation of Confidence Limit: CompareVOA (BioTools, Inc.)        analysis        Experimental:    -   Spectrometer: BioTools Chiral/R-2X FT-VCD spectrometer operated        at 4 cm⁻¹    -   Frequency Range: 2000-800 cm⁻¹    -   PEM Calibration: PEM calibrated at 1400 cm⁻¹    -   PEM Retardation Settings: PEM1=0.250*A; PEM2=0.260*λ    -   Scan Method: single 4 h scan; total #=3120×4=12480 scans) scans;        t ˜6 h.)    -   Solvent: CDCl₃    -   Concentrations: ˜10 mg/250 uL    -   Baseline Correction Method: modified half-difference (VCDE1        (corr'd)=VCDE1 minus VCDE2; VCDE2 (corr'd)=VCDE2 minus VCDE1)    -   Additional Processing: Savitsky-Golay 9-point smooth        Estimated Level of Reliability

The confidence limit in this study was estimated using CompareVOA™(BioTools, Inc.), an automated tool for quantifying the level ofagreement between two sets of spectral data.

The degree of reliability (the confidence limit) is assessed using theabsolute values of two parameters: total neighborhood similarity for theVCD correlation (TNS (VCD)) and the enantiomeric similarity index (ESI).

The degrees of reliability based on CompareVOA analysis are as follows:

*TNS (VCD) *ESI Confidence Limit (CL) Reliability (range) (range)(range) High ≧70 ≧60  >99% Medium 60-70 50-60 95-99% Low 50-60 40-5090-95% Unreliable  <50  <40  <90% *absolute valueCompareVOA Results:Spectral range: 1760-950 cm⁻¹

-   -   Region omitted: none    -   Range of statistical analysis (minimum 400 cm⁻¹): 810 cm⁻¹    -   Width of triangular weighting function: 20 cm⁻¹    -   TNS (VCD): 85.1 (absolute value)    -   ESI: 82.8 (absolute value)    -   Optimized scale factor: 0.975    -   Estimated confidence level: >99%

Intermediate 44 Methyl2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carboxylate

A solution of sodium hydrosulfite (512 mg, 2.498 mmol) in water (3.25mL) was added to a solution of methyl3-methoxy-4-(methylamino)-5-nitrobenzoate (200 mg, 0.833 mmol) and1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (167 mg,0.833 mmol) in ethanol (6.5 mL) in a microwave vial. The reactionmixture was heated in the microwave for 5 h at 100° C. The reactionmixture was diluted with DCM (20 mL), dried (Na₂SO₄), filtered andconcentrated in vacuo to afford the crude product as a colourless oil.The crude product was purified by column chromatography on a silicacartridge (25 g) using a gradient of 60% EtOAc/cyclohexane→100%EtOAc/cyclohexane. (The product eluted near the solvent front). Theappropriate fractions were combined and evaporated under vacuum to givethe product as a yellow oil—methyl2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carboxylate(260 mg, 0.666 mmol, 80% yield) LCMS (Method B): Rt=1.17 mins,MH⁺=391.3.

Intermediate 45 Methyl2-(1-(cyclopropvylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carboxylate

Prepared in a similar manner to Intermediate 44 using methyl4-(methylamino)-3-nitrobenzoate (89 mg, 0.424 mmol) and1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (85 mg,0.424 mmol).

LCMS (Method B): Rt=1.05 mins, MH⁺=361.1

Intermediate 462-(1-(Cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carboxylicacid

Methyl2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carboxylate(260 mg, 0.666 mmol) was dissolved in a 1:1 ratio of tetrahydrofuran(THF) (3.2 mL) and water (3.2 mL). To this was added lithium hydroxidemonohydrate (140 mg, 3.33 mmol) and the reaction stirred at rt for 16 h.The reaction mixture was acidified by the addition of 2M HCl (aq) (20mL) and the organics extracted into 10% MeOH/DCM (20 mL). The aqueouslayer was washed with 10% MeOH/DCM (2×20 mL) and the combined organicsdried (Na₂SO₄) and concentrated in vacuo to afford a yellow oil whichsolidified on standing (44 mg). Due to the poor recovery it was assumedthe remainder of the product remained in the aqueous layer. The aqueouslayer was further extracted with EtOAc (20 mL), DCM (2×20 mL) and 10%MeOH/DCM (8×10 mL). The combined organics were dried (Na₂SO₄) andconcentrated in vacuo. Both crude products were combined together toform2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carboxylicacid (105 mg, 0.279 mmol, 41.9% yield).

LCMS (Method B): Rt=1.00 mins, MH⁺=377.1.

Intermediate 472-(1-(Cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carboxylicacid

Prepared in a similar manner to Intermediate 46 from methyl2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carboxylate(111 mg, 0.308 mmol).

LCMS (Method B): Rt=0.90 mins, MH⁺=347.1

Intermediate 48tert-Butyl((3S,4R)-1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)-4-hydroxypiperidin-3-yl)carbamate

To a solution of2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carboxylicacid (105 mg, 0.279 mmol) in DMF (1.5 mL) was added HATU (106 mg, 0.279mmol) followed by DIPEA (0.097 mL, 0.558 mmol) and the reaction stirredat room temperature for 15 min.tert-Butyl((3S,4R)-4-hydroxypiperidin-3-yl)carbamate (60.3 mg, 0.279mmol) was added in DMF (1.5 mL) and the reaction stirred at RT for 16 h.LCMS showed complete reaction. Water (20 mL) and Et₂O (20 mL) were addedand the layers separated. The aqueous layer was extracted with furtherEt₂O (2×20 mL) and the combined organics washed with water (2×20 mL),dried (Na₂SO₄) and concentrated in vacuo to afford a yellow oil. Thecrude product was purified by flash chromatography on silica (10 g)using a gradient of DCM→100% (20% MeOH/DCM)/DCM. The appropriatefractions were combined and evaporated under vacuum to give the productas a yellowoil—tert-butyl((3S,4R)-1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)-4-hydroxypiperidin-3-yl)carbamate(146 mg, 0.254 mmol, 91% yield).

LCMS (Method B): Rt=1.03 min, MH⁺=575.3

Intermediate 49tert-butyl((3S,4R)-1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)-4-hydroxypiperidin-3-yl)carbamate

Prepared in a similar manner to Intermediate 48 from(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-4-carboxylicacid and tert-butyl((3S,4R)-4-hydroxypiperidin-3-yl)carbamate.

LCMS (Method B): Rt=0.94 mins, MH⁺=545.2.

Intermediate 1-Neopentyl-1H- Prepared in a similar manner to 50pyrrolo[2,3-b]pyridine- Intermediate 13 from 1H- 2-carbaldehydepyrrolo[2,3-b]pyridine-2- carbaldehyde and neopentyl bromide. LCMS(Method B): Rt = 1.14 mins, MH⁺ = 217.2 Intermediate1-(2-Methylbutyl)-1H- Prepared in a similar manner to 51pyrrolo[2,3-b]pyridine- Intermediate 13 from 1H- 2-carbaldehydepyrrolo[2,3-b]pyridine-2- carbaldehyde and bromo-2- methylbutane. LCMS(Method B): Rt = 1.15 mins, MH⁺ = 217.2. Intermediate 1-Isobutyl-1H-Prepared in a similar manner to 52 pyrrolo[2,3-b]pyridine- Intermediate13 from 1H- 2-carbaldehyde pyrrolo[2,3-b]pyridine-2- carbaldehyde and1-bromo-2- methylpropane. LCMS (Method B): Rt = 1.06 mins, MH⁺ = 203.1.Intermediate 1-(2-Methoxy-2- Prepared in a similar manner to 53methylpropyl)-1H- Intermediate 13 from 1H- pyrrolo[2,3-b]pyridine-pyrrolo[2,3-b]pyridine-2- 2-carbaldehyde carbaldehyde and 1-bromo-2-methoxy-2-methylpropane. LCMS (Method B): Rt = 0.92 mins, MH⁺ = 253.2Intermediate 4-Chloro-3-methoxy- Prepared in a similar manner to 545-nitrobenzoic acid Intermediate 19 from methyl 4-chloro-3-methoxy-5-nitrobenzoate. LCMS (Method B): Rt = 0.86 mins, MH⁺ =230.0 (ES⁻) Intermediate R)-tert-Butyl (1-(4- Prepared in a similarmanner to 55 chloro-3-methoxy-5- Intermediate 20 from 4-chloro-3-nitrobenzoyl)piperidin- methoxy-5-nitrobenzoic acid. 3-yl)carbamate LCMS(Method B): Rt = 1.08 mins, MH⁺ = 414.1

Intermediate 56(R)-tert-Butyl(1-(3-methoxy-4-((2-methoxyethyl)amino)-5-nitrobenzoyl)piperidin-3-yl)carbamate

2-Methoxyethylamine (0.15 mL, 1.741 mmol) was added to a stirredsolution of(R)-tert-butyl(1-(4-chloro-3-methoxy-5-nitrobenzoyl)piperidin-3-yl)carbamate(280 mg, 0.406 mmol) in N,N-dimethylformamide (DMF) (1.5 mL) at rt undernitrogen. The reaction mixture was heated to 80° C. and stirred undernitrogen overnight (16 h). LC/MS showed that the desired product hadformed with 55% purity. Water (75 mL) and diethyl ether (75 mL) wereadded to the reaction mixture and the layers separated. The aqueouslayer was further extracted with diethyl ether (2×50 mL). The organiclayers were collected, dried (Na₂SO₄), passed through a hydrophobic fritand concentrated under vacuum to afford 330 mg of crude product as anorange oil. The crude product was dissolved in a minimum volume of DCMand purified by column chromatography (25 g silica). The column waseluted with a gradient of 60-100% ethyl acetate/cyclohexane. TLC wasused to determine product fractions and the appropriate fractions werecollected and concentrated under vacuum toafford—(R)-tert-butyl(1-(3-methoxy-4-((2-methoxyethyl)amino)-5-nitrobenzoyl)piperidin-3-yl)carbamate(165.7 mg, 0.366 mmol, 90% yield) LCMS (Method B): Rt=1.04 mins,MH⁺=453.3

Intermediate (R)-tert-Butyl (1-(4- Prepared similarly to Intermediate 57(isobutylamino)-3- 56 from tert-butylamine and (R)- methoxy-5-tert-butyl (1-(4-chloro-3-methoxy-5- nitrobenzoyl)piperidin-nitrobenzoyl)piperidin-3- 3-yl)carbamate yl)carbamate. LCMS (Method B):Rt = 1.23 mins, MH⁺ = 451.3 Intermediate (S)-tert-Butyl (1-(3- Preparedin a similar manner to 58 methoxy-4- Intermediate 20 from 3-methoxy-4-(methylamino)-5- (methylamino)-5-nitrobenzoic acid.nitrobenzoyl)piperidin- LCMS (Method B): Rt = 1.02 mins, 3-yl)carbamateMH⁺ = 409.2

Intermediate 59 4-Bromo-N-methyl-2-nitro-6-(trifluoromethoxy)aniline

A solution of 4-bromo-2-nitro-6-(trifluoromethoxy)aniline (1.962 g, 6.52mmol, commercially available from, for example, Apollo Scientific) inN,N-dimethylformamide (DMF) (80 mL) was cooled with an ice/water bath to−0° C. for 10 min. Cesium carbonate (4.25 g, 13.04 mmol) was then addedand stirred, and the colour changed from yellow to red. After 10 min,methyl iodide (0.408 mL, 6.52 mmol) was added and the mixture wasallowed to return to rt with stirring under nitrogen for 3 h. LCMSshowed ˜90% conversion to the desired product with no starting materialremaining, and ˜10% formation of an impurity. The reaction mixture waspartitioned using water (400 mL) and EtOAc (400 mL), and the aqueouslayer re-extracted with EtOAc (2×400 mL). The combined organics werebackwashed with water (400 mL) and then passed through a hydrophobicfrit and concentrated in vacuo to give the crude product as a yellowoil. The sample was loaded in dichloromethane and purified on silica(Si) (100 g) using 100% cyclohexane. The appropriate fractions werecombined and evaporated in vacuo to give the required product (1.368 g,67%) as an orange solid.

LCMS (Method A): Rt=1.33 min, MH⁺=314.9

Intermediate 605-Bromo-2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-7-(trifluoromethoxy)-1H-benzo[d]imidazole

To a solution of 4-bromo-N-methyl-2-nitro-6-(trifluoromethoxy)aniline(1.368 g, 4.34 mmol) and1-ethyl-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (0.756 g, 4.34 mmol) inethanol (20 mL) was added sodium dithionite (2.67 g, 13.03 mmol) inwater (10 mL). The mixture was flushed with nitrogen and then heated to80° C. with stirring for 17 h. LCMS showed ˜52% conversion to thedesired product with no starting material remaining. The reactionmixture was partitioned between aqueous hydrochloric acid (0.25 M, 100mL) and extracted with dichloromethane (3×100 mL). The organics werecombined, dried using a hydrophobic frit and evaporated under vacuum togive the crude product as a yellow solid. The sample was loaded indichloromethane and purified by column chromatography on silica (100 g)using a gradient of 0-30% cyclohexane-ethyl acetate. The appropriatefractions were combined and evaporated in vacuo to give the requiredproduct (628 mg, 33%) as a yellow gum which solidified.

LCMS (Method A): Rt=1.46 min, MH⁺=439.1

Intermediate 61 Methyl2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-7-(trifluoromethoxy)-1H-benzo[d]imidazole-5-carboxylate

5-Bromo-2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-7-(trifluoromethoxy)-1H-benzo[d]imidazole(314 mg, 0.715 mmol), molybdenum hexacarbonyl (94 mg, 0.357 mmol),methanol (0.434 mL, 10.72 mmol), DIPEA (0.250 mL, 1.430 mmol), DMAP (175mg, 1.430 mmol) andtrans-bis(acetato)bis[o-(di-o-tolylphosphino)benzyl]dipalladium(II) (34mg, 0.036 mmol) were dissolved in 1,4-dioxane (12 mL) in a microwavevial. The reaction vessel was sealed and heated in Biotage Initiatormicrowave to 190° C. for 2 h. After allowing the reaction mixture tocool, LCMS showed ˜37% conversion to the desired product, as well as˜12% conversion to the hydrolysed product. The reaction mixture wasconcentrated in vacuo to give the crude product, methyl2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-7-(trifluoromethoxy)-1H-benzo[d]imidazole-5-carboxylate(512 mg, 1.224 mmol, 171% yield) as a brown gum which was used withoutfurther purification.

LCMS (Method A): Rt=1.32 mins, MH⁺=419.2.

Intermediate 2-(1-Ethyl-1H-pyrrolo[2,3- Prepared from methyl 2-(1- 62b]pyridin-2-yl)-1-methyl-7- ethyl-1H-pyrrolo[2,3- (trifluoromethoxy)-1H-b]pyridin-2-yl)-1-methyl-7- benzo[d]imidazole-5- (trifluoromethoxy)-1H-carboxylic acid benzo[d]imidazole-5- carboxylate in a similar manner toIntermediate 46. LCMS (Method B): Rt = 1.05 mins, MH⁺ = 405.1Intermediate (R)-tert-Butyl (1-(2-(1-ethyl- Prepared from 2-(1-ethyl-1H-63 1H-pyrrolo[2,3-b]pyridin-2- pyrrolo[2,3-b]pyridin-2-yl)-1-yl)-1-methyl-7- methyl-7-(trifluoromethoxy)- (trifluoromethoxy)-1H-1H-benzo[d]imidazole-5- benzo[d]imidazole-5- carboxylic acid (236 mg,carbonyl)piperidin-3- 0.584 mmol) and (R)-tert- yl)carbamate butylpiperidin-3-ylcarbamate in a similar manner to Intermediate 48. LCMS(Method A): Rt = 1.28 mins, MH⁺ = 587.4 Intermediate (R)-tert-Butyl(1-(7- Prepared in a similar manner 64 methoxy-1-methyl-2-(1- toIntermediate 29 from (R)- neopentyl-1H-pyrrolo[2,3- tert-butyl(1-(3-methoxy-4- b]pyridin-2-yl)-1H- (methylamino)-5-benzo[d]imidazole-5- nitrobenzoyl)piperidin-3- carbonyl)piperidin-3-yl)carbamate and 1- yl)carbamate neopentyl-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde in a Biotage Initiator microwave at 100° C.for 5 hours. LCMS (Method B): Rt = 1.23 mins, MH⁺ = 575.3. Intermediatetert-Butyl ((3R)-1-(7- Prepared in a similar manner 65methoxy-1-methyl-2-(1-(2- to Intermediate 64 from (R)- methylbutyl)-1H-tert-butyl (1-(3-methoxy-4- pyrrolo[2,3-b]pyridin-2-yl)-(methylamino)-5- 1H-benzo[d]imidazole-5- nitrobenzoyl)piperidin-3-carbonyl)piperidin-3- yl)carbamate and 1-(2- yl)carbamatemethylbutyl)-1H-pyrrolo[2,3- b]pyridine-2-carbaldehyde. LCMS (Method B):Rt = 1.23 mins, MH⁺ = 575.4 Intermediate (R)-tert-Butyl (1-(2-(1-ethyl-Prepared in a similar manner 66 1H-pyrrolo[2,3-b]pyridin-2- toIntermediate 64 from (R)- yl)-7-methoxy-1-(2- tert-butyl(1-(3-methoxy-4- methoxyethyl)-1H- ((2-methoxyethyl)amino)-5-benzo[d]imidazole-5- nitrobenzoyl)piperidin-3- carbonyl)piperidin-3-yl)carbamate and 1-ethyl-1H- yl)carbamate pyrrolo[2,3-b]pyridine-2-carbaldehyde. LCMS (Method A): Rt = 1.14 mins, MH⁺ = 577.4 Intermediate(R)-tert-Butyl (1-(2-(1-ethyl- Prepared in a similar manner 671H-pyrrolo[2,3-b]pyridin-2- to intermediate 65 from (R)-yl)-1-isobutyl-7-methoxy- tert-butyl (1-(4- 1H-benzo[d]imidazole-5-isobutylamino)-3-methoxy-5- carbonyl)piperidin-3-nitrobenzoyl)piperidin-3- yl)carbamate yl)carbamate and 1-ethyl-1H-pyrrolo[2,3-b]pyridine-2- carbaldehyde in 67% yield. LCMS (Method B): Rt= 1.22 mins, MH⁺ = 575.4 Intermediate (S)-tert-Butyl (1-(2-(1-ethyl-Prepared in a similar manner 68 1H-pyrrolo[2,3-b]pyridin-2- toIntermediate 29 from (S)- yl)-7-methoxy-1-methyl-1H- tert-butyl(1-(3-methoxy-4- benzo[d]imidazole-5- (methylamino)-5-carbonyl)piperidin-3- nitrobenzoyl)piperidin-3- yl)carbamateyl)carbamate and 1-ethyl-1H- pyrrolo[2,3-b]pyridine-2- carbaldehyde.LCMS (Method B): Rt = 1.07 mins, MH⁺ = 533.4 Intermediate (R)-tert-Butyl(1-(7- Prepared in a similar manner 69 methoxy-2-(1-(2-methoxy-2- toIntermediate 29 from (R)- methylpropyl)-1H- tert-butyl (1-(3-methoxy-4-pyrrolo[2,3-b]pyridin-2-yl)- (methylamino)-5- 1-methyl-1H-nitrobenzoyl)piperidin-3- benzo[d]imidazole-5- yl)carbamate and 1-(2-carbonyl)piperidin-3- methoxy-2-methylpropyl)-1H- yl)carbamatepyrrolo[2,3-b]pyridine-2- carbaldehyde. LCMS (Method B): Rt = 1.08 mins,MH⁺ = 591.3 Intermediate (R)-tert-Butyl (1-(2-(1- Prepared in a similarmanner 70 isobutyl-1H-pyrrolo[2,3- to Intermediate 29 from (R)-b]pyridin-2-yl)-7-methoxy- tert-butyl (1-(3-methoxy-4- 1-methyl-1H-(methylamino)-5- benzo[d]imidazole-5- nitrobenzoyl)piperidin-3-carbonyl)piperidin-3- yl)carbamate and 1-isobutyl- yl)carbamate1H-pyrrolo[2,3-b]pyridine-2- carbaldehyde. LCMS (Method B): Rt = 1.17mins, MH⁺ = 561.3.

Intermediate 71tert-Butyl((cis)-1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)-6-methylpiperidin-3-yl)carbamate(this is an Unknown Single Enantiomer with Cis-Relative Stereochemistry,Enantiomer of Intermediate 72)

Intermediate 72tert-Butyl((cis)-1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)-6-methylpiperidin-3-yl)carbamate(Enantiomer of Intermediate 71 with Cis-Relative Stereochemistry)

To a solution of2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carboxylicacid (250 mg, 0.498 mmol) in DMF (2.3 mL) was added HATU (189 mg, 0.498mmol) followed by DIPEA (0.174 mL, 0.996 mmol) and the reaction stirredat room temperature for 15 min.tert-Butyl(6-methylpiperidin-3-yl)carbamate (107 mg, 0.498 mmol) wasadded in DMF (2.3 mL) and the reaction stirred at rt for 16 h. Water (20mL) and Et₂O (20 mL) were added and the layers separated. The aqueouslayer was extracted with further Et₂O (2×20 mL) and the combinedorganics washed with water (2×20 mL), dried (Na₂SO₄) and concentrated invacuo to afford a yellow oil. The crude product was purified by flashchromatography on silica (25 g) using a gradient of DCM→100% (20%MeOH/DCM)/DCM. The appropriate fractions were combined and evaporatedunder vacuum to give the product as a yellow oil. This material waspurified further by high pH MDAP (Method E). Accordingly, the sample(160 mg) was dissolved in DMSO/MeOH (1:1, 1.8 mL) and injected in twobatches. The appropriate fractions were collected and concentrated invacuo to afford a whitesolid—tert-butyl(1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)-6-methylpiperidin-3-yl)carbamate(93 mg, 0.162 mmol, 32.6% yield). This material was sent for chiralresolution. 4 components were successfully resolved. However analysisshowed only 1-2% of the presumed minor diastereomers. The mixture wassubmitted for chiral preparative chromatography (Chiral Method D) andonly the two major components were collected:

Intermediate 71 Isomer 1:tert-butyl((cis)-1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)-6-methylpiperidin-3-yl)carbamate(50 mg, 0.087 mmol, 17.53% yield)

LCMS (Method B): Rt=1.19 min, MH⁺=573.4

Intermediate 72 Isomer 2:tert-butyl((cis)-1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)-6-methylpiperidin-3-yl)carbamate(47 mg, 0.082 mmol, 16.48% yield)

LCMS (Method B): Rt=1.19 min, MH⁺=573.4.

EXAMPLES Example 11-{[2-(1-Ethyl-1H-pyrrolo[3,2-c]pyridin-2-yl)-1-methyl-1H-benzimidazol-5-yl]carbonyl}-3-piperidinamine

TFA (0.25 mL, 3.24 mmol) was added to a stirred solution oftert-butyl(1-(2-(1-ethyl-1H-pyrrolo[3,2-c]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate(72.8 mg, 0.145 mmol) in dichloromethane (DCM) (3 mL) at roomtemperature and left stirring for 1 hour 30 minutes. The reactionmixture was concentrated under vacuum. The concentrated mixture wasdissolved in methanol loaded onto a 5 g SCX column. The column waseluted with methanol (3 CV) and then the product was eluted as a freebase with 2M ammonia in methanol (3 CV). Product fractions werecollected and concentrated under vacuum and then dried in a vacuum ovenat 40° C. to give a yellow solid (56 mg).

LCMS (Method A): Rt=0.71 min, MH⁺=403.3

Example 2A(R)-(3-Aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone

A solution of sodium hydrosulfite (353 mg, 1.722 mmol) in water (1.5 ml)was added to a solution of(R)-tert-butyl(1-(4-(methylamino)-3-nitrobenzoyl)piperidin-3-yl)carbamate(261 mg, 0.689 mmol) and1-ethyl-1H-pyrrolo[2,3-b]pyridine-2-carbaldehyde (100 mg, 0.574 mmol) inethanol (3.5 ml) in a 5 ml microwave vial. The reaction mixture washeated in the microwave for 5 hours at 100° C. Methanol was added to thereaction mixture. The reaction mixture was dried using Na₂SO₄. Thismixture was then filtered under vacuum. The crude product was loaded inDCM, onto a 50 g SNAP Si-cartridge, purified by SP4, eluting with 0-5%methanol in DCM (15 CV). The appropriate fractions were combined and thesolvent was evaporated under vacuo to give an impure product. This wasfurther purified by SP4: loaded in DCM, on a 50 g SNAP Si-cartridge,eluting with 0-5% methanol in DCM (15 CV). The appropriate fractionswere combined and the solvent was evaporated in vacuo to give theBOC-protected product. The BOC-protected product was taken up indichloromethane (DCM) (5 ml) and treated with TFA (0.663 ml, 8.61 mmol).The reaction mixture was stirred at rt for 30 min and left withoutstirring for 15 h. The reaction mixture was then concentrated underreduced pressure and the residue was loaded in methanol onto a 10 g SCXcolumn (preconditioned with MeOH). The column was washed with MeOH (3CV) and eluted with methanolic ammonia (2N) (4 CV). The methanolicammonia fractions were combined and the solvent was evaporated in vacuoto give the title compound (178 mg) as a yellow oil.

LCMS (Method B): Rt=0.63 min, MH⁺=403.2

Example 2B(R)-(3-Aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanonehydrochloride

(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone(170 mg) was taken up in MeOH (5 mL) and treated with HCl (1M in ether)(165 μL) and blown down under nitrogen to give(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride (187 mg, 0.43 mmol, 74.2% yield) as a cream solid.

LCMS (Method B): Rt=0.63 min, MH⁺=403.1

Example 3(R)-(3-Aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-c]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone

To a solution of(R)-tert-butyl(1-(2-(1-ethyl-1H-pyrrolo[2,3-c]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate(42 mg, 0.084 mmol) in dichloromethane (DCM) (1 mL) was added TFA (0.258mL, 3.34 mmol) and the reaction stirred at room temperature for 2 h. Thereaction mixture was concentrated in vacuo to afford a yellow oil. Thiswas dissolved in methanol and loaded onto an SCX cartridge (5 g). It waseluted with methanol (3 column volumes) and product eluted as free basewith 2M ammonia in methanol. The filtrate from the ammonia fractions wasconcentrated in vacuo to yield the title compound as a yellow solid (34mg).

LCMS (Method A): Rt=0.73 min, MH⁺=403.2

Example 4A(R)-(3-Aminopiperidin-1-yl)(1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone

To a stirred solution of(R)-tert-butyl(1-(1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate(1.9336 g, 3.47 mmol) in dichloromethane (DCM) (5.5 ml) was added TFA(5.05 ml, 66.0 mmol) dropwise. The reaction mixture was stirred for 45min. The mixture was concentrated in vacuo, dissolved in methanol andpurified by SPE on a pre-conditioned sulfonic acid (SCX) 70 g cartridge.The column was washed with methanol (5 CV) and the product was elutedwith a 2M Ammonia in methanol solution (4 CV). The appropriate fractionswere combined and the solvent was evaporated in vacuo to give a crudeproduct that was purified by preparative HPLC (MDAP Method E).Appropriate fractions were combined and concentrated in vacuo to yieldthe title compound (1.35 g)

LCMS (Method B): Rt=0.71 min, MH⁺=457.2

Example 4B(R)-(3-Aminopiperidin-1-yl)(1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanonehydrochloride

HCl in Et₂O (1 M) (0.15 mL, 0.15 mmol) was added dropwise to a solutionof(R)-(3-aminopiperidin-1-yl)(1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone(60 mg, 0.13 mmol) in methanol (1 mL) and diethyl ether (1 mL). Afterstirring for 2.5 h at rt, the reaction mixture was dried under a streamof nitrogen to give the required product(R)-(3-aminopiperidin-1-yl)(1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanonehydrochloride (64 mg, 0.13 mmol, 99% yield).

LCMS (Method B): Rt=0.85 min, MH⁺=457.2

Example 5A(R)-(3-Aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone

To a solution of(R)-tert-butyl(1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate(3.402 g, 6.44 mmol) in dichloromethane (DCM) (40 ml) was added TFA (9ml, 118 mmol) dropwise. The reaction mixture was stirred for 3 hours.The reaction mixture was concentrated under vacuum to afford a yellowoil. The oil was dissolved in methanol and loaded onto a 70 g SCXcartridge. The column was washed with MeOH (2 CV) and the productcollected as the free base with 2M ammonia in methanol (3 CV). Theproduct was concentrated in vacuo and dried under vacuum to afford ayellow solid. This was dissolved in hot ethanol and concentrated invacuo. It was again dissolved in hot ethanol, concentrated in vacuo anddried under vacuum to yield the title compound as a yellow solid (2.61g).

LCMS (Method A): Rt=0.89 min, MH⁺=429.3

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 8.24 (1H, d), 7.96 (1H, d), 7.51-7.62(2H, m), 7.21 (1H, d), 7.05 (1H, dd), 6.96 (1H, s), 4.41 (2H, d), 3.82(3H, s), 3.28-4.26 (2H, m), 2.40-2.66 (1H, m), 2.37-2.64 (2H, m),1.62-1.77 (1H, m), 1.15-1.60 (4H, m), 0.92-1.15 (2H, m), 0.07-0.16 (2H,m), 0.03-0.04 (2H, m)

Example 5B(R)-(3-Aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride

To a solution of(R)-(3-aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone(2.61 g, 6.09 mmol) in dichloromethane (DCM) (1.5 mL) was added HCl (2Min diethyl ether) (3 ml, 6.00 mmol). The mixture was sonicated for 2minutes and then concentrated under vacuum to afford a yellow solid.This was dissolved in a minimum volume of hot ethanol. The solvent wasremoved under nitrogen and the product dried in a vacuum pistol at 50°C. overnight and then at 60° C. over the weekend to give the titlecompound as a yellow solid (2.7 g).

LCMS (Method A): Rt=0.88 min, MH⁺=429.3

Example 6(R)-(3-Aminopiperidin-1-yl)(2-(1-ethyl-5-methoxy-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone

To a stirred solution of(R)-tert-butyl(1-(2-(1-ethyl-5-methoxy-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate(24 mg, 0.045 mmol) in DCM (2 mL) was added TFA (2 mL, 26.0 mmol)dropwise with continuous stirring. The reaction was stirred at roomtemperature under nitrogen for 1 h. The reaction mixture wasconcentrated in vacuo before being dissolved in MeOH and purified by SPEon sulfonic acid (SCX) 1 g, first washing with MeOH and then elutingusing a 10% NH₃/MeOH solution to give the free base of the product. Theappropriate fractions were combined and evaporated in vacuo before beingazeotroped with cyclohexane to give the title compound as a pale yellowsolid (13 mg).

LCMS (Method A): Rt=0.84 min, MH⁺=433.3

Example 7(R)-(3-Aminopiperidin-1-yl)(2-(1-ethyl-5-methoxy-1H-pyrrolo[2,3-c]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone

To a stirred solution of (R)-tert-butyl(1-(2-(1-ethyl-5-methoxy-1H-pyrrolo[2,3-c]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate(106 mg, 0.199 mmol) in dichloromethane (DCM) (3 mL) was added TFA (3mL, 38.9 mmol) dropwise with continuous stirring. The reaction wasstirred at room temperature under nitrogen for 1 hr. LCMS showed thereaction was complete with no starting material remaining. The reactionmixture was concentrated in vacuo before being dissolved in MeOH andpurified by SPE on sulfonic acid (SCX) 5 g, first washing with MeOH andthen eluting using a 10% NH₃/MeOH solution to give the free base of theproduct. The appropriate fractions were combined and evaporated in vacuobefore being azeotroped with cHex and dried on the high vacuum line togive the required product (69 mg) as a white solid.

LCMS (Method A): Rt=0.80 min, MH⁺=433.2

Example 82-(5-{[(3R)-3-Amino-1-piperidinyl]carbonyl}-1-methyl-1H-benzimidazol-2-yl)-1-ethyl-1H-pyrrolo[2,3-b]pyridin-5-ol

A solution of(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-5-methoxy-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone(11 mg, 0.025 mmol) in dichloromethane (DCM) (3 mL) was cooled to −0° C.using an ice-water bath under nitrogen. Boron tribromide (8 μL, 0.085mmol) was added to the reaction dropwise with vigorous stirring. Thereaction mixture was allowed to return to room temperature withstirring, over 4 hours. The reaction mixture was partitioned with water(5 mL), the organic layer was isolated using a hydrophobic frit, and theaqueous layer was re-extracted with DCM (2×10 mL). The combined organiclayers were evaporated in vacuo but LCMS showed there was no product.The aqueous layer was neutralised by the dropwise addition of NaHCO₃,partitioned with DCM and separated. The aqueous was re-extracted withDCM (2×15 mL) and the combined organic layers were concentrated in vacuoto give the crude product. The residue was dissolved in DMSO 1 mL andpurified by MDAP with an ammonium bicarbonate modifier (Method E). Theappropriate fraction was evaporated in vacuo to give the title compoundas a yellow gum (12 mg).

LCMS (Method A): Rt=0.67 min, MH⁺=419.25

Example 9A(R)-(3-Aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone

To a solution of(R)-tert-butyl(1-(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate(4.4 g, 8.26 mmol) in dichloromethane (DCM) (20 ml) was added TFA (9 ml,118 mmol) dropwise. The reaction mixture was stirred for 1 hour 30minutes. LC/MS showed that that desired product had formed with nostarting material remaining. The reaction mixture was concentrated undervacuum to afford an oil. The oil was dissolved in methanol and splitinto two equal batches and passed through two separate 70 g SCXcartridges. The columns were washed with MeOH (2 CV) and the productcollected from both columns as the free base with 2M ammonia in methanol(3 CV). The product was concentrated under vacuo and dried under vacuumto afford the title compound as a white solid (3.46 g).

LCMS (Method A): Rt=0.89 min, MH⁺=433.4

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 8.42 (dd, 1H), 8.12 (dd, 1H), 7.32 (s,1H), 7.22 (dd, 1H), 7.06 (s, 1H), 6.87 (s, 1H), 4.62 (q, 2H), 4.14 (s,3H), 3.99 (s, 3H), 3.50-4.43 (m, 2H), 2.63-2.71 (m, 1H), 2.58-3.11 (m,2H), 1.82-1.91 (m, 1H), 1.61-1.76 (m, 1H), 1.52-1.59 (m, 2H), 1.39-1.50(m, 1H), 1.25 (t, 3H), 1.20-1.26 (m, 1H)

Example 9B(R)-(3-Aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride

(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone(2.365 g) was dissolved in DCM (6 ml) and HCl (2M in diethyl ether)(2.735 ml, 5.47 mmol) was added to the solution. The solvent was thenremoved under nitrogen and concentrated under vacuum to afford the titlecompound as a white solid (2.43 g).

LCMS (Method A): Rt=0.89 min, MH⁺=433.3

Example 10A(R)-(3-Aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone

To a solution of(R)-tert-butyl(1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate(1.5587 g, 2.79 mmol) in DCM (10 ml) was added TFA (5 mL, 65.3 mmol)dropwise. The reaction mixture was stirred for 30 minutes and thenconcentrated under vacuum to afford an oil. The oil was dissolved inmethanol and loaded onto a 70 g SCX cartridge. The column was washedwith MeOH (2 CV) and the product collected as the free base with 2Mammonia in methanol (3 CV). The product was concentrated in vacuo yielda crude product. This was purified by high pH MDAP (Method E).Appropriate fractions were combined and concentrated in vacuo to yieldthe title compound as a white solid, 1.127 g.

LCMS (Method A): Rt=0.96 min, MH⁺=459.3

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 8.27 (1H, dd), 7.99 (1H, dd), 7.22 (1H,s), 7.08 (1H, dd), 6.94 (1H, s), 6.76 (1H, s), 4.36 (2H, d), 4.00 (3H,s), 3.86 (3H, s), 3.45-4.27 (4H, m), 2.80-2.97 (1H, m), 2.64-2.80 (2H,m), 1.71-1.87 (1H, m), 1.50-1.66 (1H, m), 1.28-1.44 (1H, m), 1.14-1.29(1H, m), 0.84-1.07 (1H, m), 0.08-0.22 (2H, m), −0.08-0.05 (2H, m)

Example 10B(R)-(3-Aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride

(R)-(3-Aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone(2.2 g) was dissolved in a minimum volume of DCM and HCl (2M in diethylether) (2.4 ml, 4.80 mmol) added to the solution. The resultingsuspension was sonicated for 2 minutes and the solution concentratedunder vacuum to afford the title compound as a white solid (2.57 g)

LCMS (Method A): Rt=0.95 min, MH⁺=459.3

Example 11A(R)-(3-Aminopiperidin-1-yl)(7-methoxy-1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone

To a solution of(R)-tert-butyl(1-(7-methoxy-1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate(4.31 g, 7.35 mmol) in dichloromethane (DCM) (20 ml) at 00° C. was addedTFA (9 ml, 118 mmol) dropwise. The reaction mixture was stirred for 5minutes and allowed to warm to room temperature and stirred for 3 hours.The reaction mixture was concentrated under vacuum to afford an oil. Theoil was dissolved in methanol and split into two equal batches. Thesewere passed through two separate 70 g SCX cartridges. The columns werewashed with MeOH (2 CV) and the product collected from both columns asthe free base with 2M ammonia in methanol (3 CV). The product wasconcentrated and dried under vacuum to afford the title compound as awhite solid (2.53 g).

LCMS (Method A): Rt=0.97 min, MH⁺=487.1

¹H NMR (400 MHz, DMSO-d₆) δ ppm: 8.48 (dd, 1H), 8.21 (dd, 1H), 7.33 (s,1H), 7.32-7.33 (m, 1H), 7.31 (s, 1H), 6.88 (s, 1H), 5.74 (q, 2H), 4.19(s, 3H), 4.00 (s, 3H), 3.51-4.39 (m, 2H), 2.64-2.73 (m, 1H), 2.62-3.01(m, 2H), 1.83-1.90 (m, 1H), 1.62-1.77 (m, 1H), 1.49-1.59 (m, 2H),1.39-1.50 (m, 1H), 1.17-1.30 (m, 1H)

Example 11B(R)-(3-Aminopiperidin-1-yl)(7-methoxy-1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-bl]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride

(R)-(3-Aminopiperidin-1-yl)(7-methoxy-1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone(1.5073 g) was dissolved in DCM (5 ml) and HCl (2M in diethyl ether)(1.5 ml, 3.00 mmol) was added to the solution. The solvent was thenremoved under nitrogen and concentrated under vacuum to afford the titlecompound as a white solid (1.61 g)

LCMS (Method A): Rt=0.96 min, MH⁺=487.2

Example 12(3-Aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone

To a solution oftert-butyl(1-(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate(104 mg, 0.207 mmol) in dichloromethane (DCM) (1 mL) was added TFA(0.367 mL, 4.76 mmol) and the reaction stirred at room temperature for 2h. LCMS (A1) showed no desired product but reaction had progressed to 1major product. The reaction mixture was concentrated in vacuo to afforda colourless oil. This was dissolved in methanol and loaded onto an SCXcartridge (5 g). It was eluted with methanol (3 column volumes) andproduct eluted as free base with 2M ammonia in methanol. The filtratefrom the ammonia fractions was concentrated in vacuo to yield a yellowsolid—(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone(81 mg, 0.201 mmol, 97% yield).

LCMS (Method B): Rt=0.64 min, MH⁺=403.2

Example 13((3S,4R)-3-Amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride

To a flask containingtert-butyl((3S,4R)-1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazole-5-carbonyl)-4-hydroxypiperidin-3-yl)carbamate(37 mg, 0.068 mmol) in dichloromethane (DCM) (1 mL) was added TFA (0.199mL, 2.58 mmol) and the reaction was stirred for 1 h. The reactionmixture was concentrated in vacuo to afford a brown oil. This wasdissolved in methanol and loaded onto an SCX cartridge (5 g). It waseluted with methanol (3 column volumes) and product eluted as free basewith 2M ammonia in methanol. The filtrate from the ammonia fractions wasconcentrated in vacuo to yield a yellowoil—((3S,4R)-3-amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone(32 mg, 0.068 mmol, 100% yield). This was dissolved in dichloromethane(DCM) (1 mL) in a vial and HCl (2 M in Et₂O) (0.034 mL, 0.068 mmol) wasadded. The resultant suspension was sonicated for 5 min and allowed tostand for 15 min. The solvent was then removed under a positive pressureof nitrogen and the product dried in vacuo to afford((3S,4R)-3-amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride (32 mg, 0.067 mmol, 98% yield) as a white solid.

LCMS (Method A): Rt=0.83 mins, MH⁺=445.3

Example 14((3S,4R)-3-Amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride

To a flask containingtert-butyl((3S,4R)-1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)-4-hydroxypiperidin-3-yl)carbamate(143 mg, 0.249 mmol) in dichloromethane (DCM) (1.5 mL) was added TFA(0.307 mL, 3.98 mmol) and the reaction was stirred for 1 h. LCMS showedcomplete reaction. The reaction mixture was concentrated in vacuo toafford a brown oil. This was dissolved in methanol and loaded onto anSCX cartridge (5 g). It was eluted with methanol (3 column volumes) andproduct eluted as free base with 2M ammonia in methanol. The filtratefrom the ammonia fractions was concentrated in vacuo to yield a yellowoil—97% purity by LCMS. The crude product (104 mg) was taken up inDMSO/MeOH (1:1, 1.8 mL) and further purified by MDAP (Method E, 2injections). The appropriate fractions were combined and concentrated invacuo to afford the desired product as a colourlessoil—((3S,4R)-3-amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone(71 mg, 0.150 mmol, 60.1% yield). The free base (71 mg) was dissolved indichloromethane (DCM) (1 mL) in a vial and HCl (2M in Et₂O) (0.075 mL,0.15 mmol) was added. The resultant suspension was sonicated for 5 minand allowed to stand for 15 min. The solvent was then removed under apositive pressure of nitrogen and the product dried in vacuo to afford((3S,4R)-3-amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride (78 mg, 0.153 mmol, 61.3% yield) as an off white solid.

LCMS (Method A): Rt=0.74 min, MH⁺=475.3

1H NMR (600 MHz, DMSO-d6) δ ppm 8.41 (dd, J=4.6, 1.7 Hz, 1H), 8.13 (dd,J=7.9, 1.7 Hz, 1H), 7.94-8.11 (m, 3H), 7.44 (s, 1H), 7.22 (dd, J=7.9,4.6 Hz, 1H), 7.08 (s, 1H) 6.96 (s, 1H), 5.71 (br. s., 1H), 4.50 (d,J=6.9 Hz, 2H), 4.14 (s, 3H), 4.05-4.10 (m, 1H), 4.00 (s, 3H), 3.79-3.99(m, 1H), 3.40-3.62 (m, 3H), 3.28-3.35 (m, 1H), 1.68-1.86 (m, 2H)1.07-1.18 (m, 1H), 0.24-0.35 (m, 2H), 0.08-0.18 (m, 2H).

Example 15: (R)-(3- Aminopiperidin-1-yl)(2- (1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl- 7-(trifluoromethoxy)-1H- benzo[d]imidazol-5-yl)methanone

Prepared in a similar manner to Example 1 from (R)-tert-butyl(1-(2-(1-ethyl-1H-pyrrolo[2,3- b]pyridin-2-yl)-1-methyl-7-(trifluoromethoxy)-1H- benzo[d]imidazole-5- carbonyl)piperidin-3-yl)carbamate. LCMS (Method A): Rt = 1.01 mins, MH⁺ = 487.2 Example 16:(R)-(3- Aminopiperidin-1-yl)(7- methoxy-1-methyl-2-(1- neopentyl-1H-pyrrolo[2,3-b]pyridin-2- yl)-1H-benzo[d]imidazol- 5-yl)methanone

Prepared in a similar manner to Example 1 from (R)-tert-butyl(1-(7-methoxy-1-methyl-2-(1- neopentyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H- benzo[d]imidazole-5- carbonyl)piperidin-3-yl)carbamate. LCMS (Method A): Rt = 1.06 mins, MH⁺ = 475.3 Example 17:((R)-3- Aminopiperidin-1-yl)(7- methoxy-1-methyl-2-(1-(2-methylbutyl)-1H- pyrrolo[2,3-b]pyridin-2- yl)-1H-benzo[d]imidazol-5-yl)methanone

Prepared in a similar manner to Example 1from tert-butyl ((3R)-1-(7-methoxy-1-methyl-2-(1-(2- methylbutyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H- benzo[d]imidazole-5- carbonyl)piperidin-3-yl)carbamate. LCMS (Method A): Rt = 1.04 mins, MH⁺ = 475.3

Example 18(R)-(3-Aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride

To a solution of(R)-tert-butyl(1-(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazole-5-carbonyl)piperidin-3-yl)carbamate(110 mg, 0.191 mmol) in dichloromethane (DCM) (2 mL) was added TFA (0.35mL, 4.57 mmol). The reaction mixture was stirred for 40 minutes. LCMSshowed that the desired product had formed with 98% purity. The reactionmixture was concentrated under vacuum to afford a yellow oil. The oilwas dissolved in methanol and loaded onto a SCX cartridge (10 g). Thecolumn was washed with MeOH (3 CV) and the product collected as the freebase with 2M ammonia in methanol (8 CV). The product was concentrated invacuo to afford a colourless oil. The product was dissolved in of 1:1DMSO/MeOH (1.8 mL) and two 0.9 mL samples were purified by MDAP (MethodE). Product fractions were collected and concentrated under vacuum toafford(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-5-yl)methanoneas a colourless oil. The colourless oil was dissolved in dichloromethane(DCM) (2 mL) and transferred to a vial and HCl (2M in diethyl ether)(0.06 mL, 0.120 mmol) was added to the solution. The solvent was removedunder nitrogen and then the sample dried in a vacuum pistol overnight toafford(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride (75.8 mg, 0.148 mmol, 77% yield)—as a white solid.

LCMS (Method A): Rt=0.88 mins, MH⁺=477.4

Example 19: (S)-(3- Aminopiperidin-1-yl)(2-(1- ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1- methyl-1H- benzo[d]imidazol-5-yl)methanone, hydrochloride

Prepared in a similar manner to Example 13 from (S)-tert-butyl(1-(2-(1-ethyl- 1H-pyrrolo[2,3-b]pyridin-2- yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5- carbonyl)piperidin-3- yl)carbamate. LCMS (MethodA): Rt = 0.90 mins, MH⁺ = 433.3 Example 20: (R)-(3-Aminopiperidin-1-yl)(2-(1- ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-isobutyl-7- methoxy-1H- benzo[d]imidazol-5-yl)methanone, hydrochloride

Prepared in a similar manner to Example 13 from (R)-tert-butyl(1-(2-(1-ethyl- 1H-pyrrolo[2,3-b]pyridin-2- yl)-1-isobutyl-7-methoxy-1H-benzo[d]imidazole-5- carbonyl)piperidin-3- yl)carbamate. LCMS (MethodA): Rt = 0.90 mins, MH⁺ = 433.3 Example 21: (R)-(3-Aminopiperidin-1-yl)(7- methoxy-2-(1-(2-methoxy-2- methylpropyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1- methyl-1H- benzo[d]imidazol-5-yl)methanone

Prepared in a similar manner to Example 1 from (R)-tert-Butyl(1-(7-methoxy- 2-(1-(2-methoxy-2- methylpropyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1- methyl-1H- benzo[d]imidazole-5-carbonyl)piperidin-3- yl)carbamate. LCMS (Method B): Rt = 0.74 mins, MH⁺= 491.3 Example 22: (R)-(3- Aminopiperidin-1-yl)(2-(1-isobutyl-1H-pyrrolo[2,3- b]pyridin-2-yl)-7-methoxy-1- methyl-1H-benzo[d]imidazol-5- yl)methanone, hydrochloride

Prepared in a similar manner to Example 18 from (R)-tert-Butyl (1-(2-(1-isobutyl-1H-pyrrolo[2,3- b]pyridin-2-yl)-7-methoxy-1- methyl-1H-benzo[d]imidazole-5- carbonyl)piperidin-3- yl)carbamate. LCMS (MethodB): Rt = 0.76 mins, MH⁺ = 461.2

Example 23((cis)-5-Amino-2-methylpiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride (enantiomer of Example 24 with cis-relativestereochemistry)

To a flask containingtert-butyl((3S,6R)-1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)-6-methylpiperidin-3-yl)carbamate(47 mg, 0.082 mmol) in dichloromethane (DCM) (1 mL) was added TFA (0.253mL, 3.28 mmol) and the reaction was stirred for 2.5 h. The reactionmixture was concentrated in vacuo to afford a brown oil. This wasdissolved in methanol and loaded onto an SCX cartridge (5 g). It waseluted with methanol (3 column volumes) and product eluted as free basewith 2M ammonia in methanol. The filtrate from the ammonia fractions wasconcentrated in vacuo to yield a yellowoil—((2R,5S)-5-amino-2-methylpiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone(35 mg, 0.074 mmol, 90% yield). The free base (35 mg) was dissolved indichloromethane (DCM) (1 mL) in a vial and HCl (2M in Et₂O) (0.037 mL,0.074 mmol) was added. The resultant suspension was sonicated for 5 minand allowed to stand for 15 min. The solvent was then removed under apositive pressure of nitrogen and the product dried in vacuo to afford((2R,5S)-5-amino-2-methylpiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride (37 mg, 0.073 mmol, 89% yield) as a beige solid.

LCMS (Method A): Rt=0.98 min, MH⁺=473.3

Example 24((cis)-5-Amino-2-methylpiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone,hydrochloride (Enantiomer of Example 23 with Cis-RelativeStereochemistry)

Prepared in a similar manner to Example 23 fromtert-butyl((3S,6R)-1-(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazole-5-carbonyl)-6-methylpiperidin-3-yl)carbamate.

LCMS (Method A): Rt=0.98 min, MH⁺=473.3

Biological Data

PAD4 Enzyme Expression

Recombinant human PAD4 (residues 1-663) was expressed in E. coli as anN-terminal GST-tagged fusion protein. During purification of theprotein, the GST tag was removed by cleavage with PreScission Protease(GE Healthcare). Activity of the final product was determined using aFLINT NH₃ release assay.

PAD4 Enzyme Assay: Conditions A

8 μl of PAD4 enzyme diluted to an assay concentration of 75 nm in assaybuffer (a).: (100 mM HEPES, 50 mM NaCl, 2 mM DTT and 0.6 mg/ml BSA pH8), or in assay buffer (b).: (100 mM HEPES, 50 mM NaCl, 2 mM DTT, 7.5%glycerol and 1.5 mM CHAPS pH 8), and added to wells containing 0.1 μl ofvarious concentrations of compound or DMSO vehicle (0.8% final) in aGreiner high volume 384 well black plate. Following 30 minspre-incubation at room temperature, the reaction was initiated by theaddition of 4 μl of substrate buffer containing 3 mMN-a-benzoyl-L-arginine ethyl ester (BAEE), 100 mM HEPES, 50 mM NaCl, 600uM CaCl₂ (2H₂O) and 2 mM DTT, pH 8.0. The reaction was stopped after 100mins with the addition of 38 μl stop/detection buffer containing 50 mMEDTA, 2.6 mM phthalaldehyde and 2.6 mM DTT. Assay incubated at roomtemperature for 90 mins before measuring fluorescent signal (λ_(ex)413/λ_(em) 476) on an Envision plate reader (Perkin Elmer Life Sciences,Waltham, Mass., USA)

PAD4 Enzyme Assay: Conditions B

8 μl of PAD4 enzyme diluted to an assay concentration of 30 nM in assaybuffer (100 mM HEPES, 50 mM NaCl, 2 mM DTT and 0.6 mg/ml BSA pH 8), andadded to wells containing 0.1 μl of various concentrations of compoundor DMSO vehicle (0.8% final) in a Greiner high volume 384 well blackplate. Following 30 mins pre-incubation at room temperature, thereaction was initiated by the addition of 4 μl of substrate buffercontaining 3 mM N-a-benzoyl-L-arginine ethyl ester (BAEE), 100 mM HEPES,50 mM NaCl, 600 uM CaC₂ (2H₂O) and 2 mM DTT, pH 8.0. The reaction wasstopped after 60 mins with the addition of 38 μl stop/detection buffercontaining 50 mM EDTA, 2.6 mM phthalaldehyde and 2.6 mM DTT. Assayincubated at room temperature for 90 mins before measuring fluorescentsignal (λ_(ex) 405/λ_(em) 460) on an Envision plate reader (Perkin ElmerLife Sciences, Waltham, Mass., USA)

PAD2 Enzyme Expression

Recombinant human PAD2 (residues 1-665) was expressed inbaculovirus-infected Sf9 insect cells as an N-terminal 6His-FLAG-taggedfusion protein. Activity of the final product was determined using aFLINT NH₃ release assay.

PAD2 Enzyme Assay

8 μl of PAD2 enzyme diluted to an assay concentration of 30 nM in assaybuffer (100 mM HEPES, 50 mM NaCl, 2 mM DTT, 7.5% glycerol and 1.5 mMCHAPS pH 8), and added to wells containing 0.1 μl of variousconcentrations of compound or DMSO vehicle (0.8% final) in a Greinerhigh volume 384 well black plate. Following 30 mins pre-incubation atroom temperature, the reaction was initiated by the addition of 4 μl ofsubstrate buffer containing 180 uM N-a-benzoyl-L-arginine ethyl ester(BAEE), 100 mM HEPES, 50 mM NaCl, 240 uM CaCl₂ (2H₂O) and 2 mM DTT, pH8.0. The reaction was stopped after 90 mins with the addition of 38 μlstop/detection buffer containing 50 mM EDTA, 2.6 mM phthalaldehyde and2.6 mM DTT. Assay incubated at room temperature for 90 mins beforemeasuring fluorescent signal (λ_(ex) 405/λ_(em) 460) on an Envisionplate reader (Perkin Elmer Life Sciences, Waltham, Mass., USA)

Results

Examples 1, 2A, 2B, 3, 4A, 4B, 5B, 6, 7, 8, 9A, 9B, 10B, 11A, 11B, 12,13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23 and 24 were tested in thePAD4 enzyme assay above or similar assays and had a mean pIC₅₀ in therange of 5 to 7.5. The mean pIC₅₀ for Example 5B was 6.7; Example 9B,mean pIC₅₀ was 6.7; Example 10B, mean pIC₅₀ was 7.3; Example 11B, meanpIC₅₀ was 6.9; for Example 14, mean pIC₅₀ was 7.1.

To assess selectivity for PAD4 over PAD2, the following examples 2B, 5B,9B, 10B, 11B, 13, 16, 19 and 22 were tested in the PAD2 enzyme assayabove or similar assays and had a mean pIC₅₀ in the range of <4.1 to4.2. The mean pIC₅₀ values for Examples 5B, 9B, 10B, 11B, and 14 wereall <4.1.

The invention claimed is:
 1. A method of treating rheumatoid arthritiscomprises administering to a patient in need thereof a PAD4 inhibitoryeffective amount of a compound of formula I:

or a pharmaceutically acceptable salt thereof, wherein: R₁ is hydrogenor C₁₋₆alkyl; R₂ is hydrogen, C₁₋₆alkyl, perhalomethylC₀₋₅alkyl-O—, orC₁₋₆alkoxy; R₃ is hydrogen, C₁₋₆alkyl, or C₁₋₆alkoxyC₁₋₆alkyl; R₄ ishydrogen, C₁₋₆alkyl, halomethylC₁₋₆alkyl; or unsubstitutedC₃₋₆cycloalkylC₁₋₆alkyl; A is C—R₅ or N; B is C—R₆ or N; D is C—R₇ or N;with the proviso that at least one of A, B, and D, is N; R₅ is hydrogenor C₁₋₆alkyl; R₆ is hydrogen or C₁₋₆alkyl; R₇ is hydrogen, C₁₋₆alkyl,C₁₋₆alkoxy, or hydroxy; R₈ is hydrogen or C₁₋₆alkyl, with the provisothat one of R₄ and R₈ is hydrogen; R₉ is hydrogen or hydroxy; and R₁₀ ishydrogen or C₁₋₆alkyl; or a pharmaceutically acceptable compositioncomprising said compound of formula I.
 2. The method according to claim1, wherein said method comprises administering to said patient apharmaceutically acceptable composition comprising said compound offormula I.
 3. The method according to claim 2, wherein: R₁ is hydrogen;R₂ is hydrogen, C₁₋₆alkyl or C₁₋₆alkoxy; R₃ is C₁₋₆alkyl; and R₄ isC₁₋₆alkyl, unsubstituted C3-6cycloalkylC₁₋₆alkyl, orperhalomethylC₁₋₆alkyl.
 4. The method according to claim 3, wherein: R₅is hydrogen; R₆ is hydrogen; R₇ is hydrogen, C₁₋₆alkoxy, or hydroxyl;and R₈ is hydrogen.
 5. The method according to claim 4, wherein R₉ ishydrogen.
 6. The method according to claim 4, wherein R₉ is hydroxy. 7.The method according to claim 4, wherein R₁₀ is hydrogen.
 8. The methodaccording to claim 4, wherein R₁₀ is C₁₋₆alkyl.
 9. The method accordingto claim 2, wherein said compound, or pharmaceutically acceptable saltthereof, is selected from the group consisting of:1-{[2-(1-ethyl-1H-pyrrolo[3,2-c]pyridin-2-yl)-1-methyl-1H-benzimidazol-5-yl]carbonyl}-3-piperidinamine;(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-c]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;(R)-(3-aminopiperidin-1-yl)(1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone;(R)-(3-aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-5-methoxy-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-5-methoxy-1H-pyrrolo[2,3-c]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;2-(5-{[(3R)-3-amino-1-piperidinyl]carbonyl}-1-methyl-1H-benzimidazol-2-yl)-1-ethyl-1H-pyrrolo[2,3-b]pyridin-5-ol;(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;(R)-(3-aminopiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;(R)-(3-aminopiperidin-1-yl)(7-methoxy-1-methyl-2-(1-(2,2,2-trifluoroethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone;(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;((3S,4R)-3-amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;((3S,4R)-3-amino-4-hydroxypiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-7-(trifluoromethoxy)-1H-benzo[d]imidazol-5-yl)methanone;(R)-(3-aminopiperidin-1-yl)(7-methoxy-1-methyl-2-(1-neopentyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone;((R)-3-aminopiperidin-1-yl)(7-methoxy-1-methyl-2-(1-(2-methylbutyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1H-benzo[d]imidazol-5-yl)methanone;(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-(2-methoxyethyl)-1H-benzo[d]imidazol-5-yl)methanone;(S)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;(R)-(3-aminopiperidin-1-yl)(2-(1-ethyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-isobutyl-7-methoxy-1H-benzo[d]imidazol-5-yl)methanone;(R)-(3-aminopiperidin-1-yl)(7-methoxy-2-(1-(2-methoxy-2-methylpropyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;(R)-(3-aminopiperidin-1-yl)(2-(isobutyl-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone;and((cis)-5-amino-2-methylpiperidin-1-yl)(2-(1-(cyclopropylmethyl)-1H-pyrrolo[2,3-b]pyridin-2-yl)-7-methoxy-1-methyl-1H-benzo[d]imidazol-5-yl)methanone.